49 Matching Annotations
  1. Oct 2024
    1. Author response:

      The following is the authors’ response to the original reviews.

      eLife assessment This valuable paper reports a theoretical framework and methodology for identifying Cancer Driving Nucleotides (CDNs), primarily based on single nucleotide variant (SNV) frequencies. A variety of solid approaches indicate that a mutation recurring three or more times is more likely to reflect selection rather than being the consequence of a mutation hotspot. The method is rigorously quantitative, though the requirement for larger datasets to fully identify all CDNs remains a noted limitation. The work will be of broad interest to cancer geneticists and evolutionary biologists. 

      The key criticism “the requirement for larger datasets to fully identify all CDNs remains a noted limitation” that is also found in both reviews. We have clarified the issue in the main text, the relevant parts, from which are copied below. The response below also addresses many comments in the reviews. In addition, Discussion of eLife-RP-RA-2024-99341 has been substantially expanded to answer the questions of Reviewer 2.

      We shall answer the boldface comment in three ways. First, it can be answered using GENIE data. Fig. 7 of the main text (eLife-RP-RA-2024-99340) shows that, when n increases from ~ 1000 to ~ 9,000, the numbers of discovered CDNs increase by 3 – 5 fold, most of which come from the two-hit class. Hence, the power of discovering more CDNs with larger datasets is evident. By extrapolation, a sample size of 100,000 should be able to yield 90% of all CDNs, as calculated here. (Fig. 7 also addresses the queries of whether we have used datasets other than TCGA. We indeed have used all public data, including GENIE and COSMIC.) 

      Second, the power of discovering more cancer driver genes by our theory is evident even without using larger datasets. Table 3 of the companion study (eLife-RP-RA-2024-99341) shows that, averaged across cancer types, the conventional method would identify 45 CDGs while the CDN method tallies 258 CDGs. The power of the CDN method is demonstrated. This is because the conventional approach has to identify CDGs (cancer driver genes) in order to identify the CDNs they carry. However, many CDNs occur in non-CDGs and are thus missed by the conventional approach. In Supplementary File S2, we have included a full list of CDNs discovered in our study, along with population allele frequency annotations from gnomAD. The distribution patterns of these CDNs across different cancer types show their pan-cancer properties as further explored in the companion paper.

      Third, while many, or even most CDNs occur in non-CDGs and are thus missed, the conventional approach also includes non-CDN mutations in CDGs. This is illustrated in Fig. 5 of the companion study (eLife-RP-RA-2024-99341) that shows the adverse effect of misidentifications of CDNs by the conventional approach. In that analysis, the gene-targeting therapy is effective if the patient has the CDN mutations on EGFR, but the effect is reversed if the EGFR mutations are non-CDN mutations.

      Reviewer #1 (Public Review):

      The authors developed a rigorous methodology for identifying all Cancer Driving Nucleotides (CDNs) by leveraging the concept of massively repeated evolution in cancer. By focusing on mutations that recur frequently in pan-cancer, they aimed to differentiate between true driver mutations and neutral mutations, ultimately enhancing the understanding of the mutational landscape that drives tumorigenesis. Their goal was to call a comprehensive catalogue of CDNs to inform more effective targeted therapies and address issues such as drug resistance.

      Strengths

      (1) The authors introduced a concept of using massively repeated evolution to identify CDNs. This approach recognizes that advantageous mutations recur frequently (at least 3 times) across cancer patients, providing a lens to identify true cancer drivers.

      (2) The theory showed the feasibility of identifying almost all CDNs if the number of sequenced patients increases to 100,000 for each cancer type.

      Weaknesses

      (1) The methodology remains theoretical and no novel true driver mutations were identified in this study.

      We now address the weakness criticism, which is gratefully received.

      The second part of the criticism (no novel true driver mutations were identified in this study) has been answered in the long responses to eLife assessment above. The first part “The methodology remains theoretical” is somewhat unclear. It might be the lead to the second part. However, just in case, we interpret the word “theoretical” to mean “the lack of experimental proof” and answer below.

      As Reviewer #1 noted, a common limitation of theoretical and statistical analyses of cancer drivers is the need to validate their selective advantage through in vitro or in vivo functional testing. This concern is echoed by both reviewers in the companion paper (eLife-RP-RA-2024-99341), prompting us to consider the methodology for functional testing of potential cancer drivers. An intuitive approach would involve introducing putative driver mutations into normal cells and observing phenotypic transformation in vitro and in vivo. In a recent stepwise-edited human melanoma model, Hodis et al. demonstrated that disease-relevant phenotypes depend on the “correct” combinations of multiple driver mutations (Hodis et al. 2022). Other high-throughput strategies can be broadly categorized into two approaches: (1) introducing candidate driver mutations into pre-malignant model systems that already harbor a canonical mutant driver (Drost and Clevers 2018; Grzeskowiak et al. 2018; Michels et al. 2020) and (2) introducing candidate driver mutations into growth factor-dependent cell models and assessing their impact on resulting fitness (Bailey et al. 2018; Ng et al. 2018). The underlying assumption of these strategies is that the fitness outcomes of candidate driver mutations are influenced by pre-existing driver mutations and the specific pathways or cancer hallmarks being investigated. This confines the functional test of potential cancer driver mutations to conventional cancer pathways. A comprehensive identification of CDNs is therefore crucial to overcome these limitations. In conjunction with other driver signal detection methods, our study aims to provide a more comprehensive profile of driver mutations, thereby enabling the functional testing of drivers involved in non-conventional cancer evolution pathways.

      (2) Different cancer types have unique mutational landscapes. The methodology, while robust, might face challenges in uniformly identifying CDNs across various cancers with distinct genetic and epigenetic contexts.

      We appreciate the comment. Indeed, different cancer types should have different genetic and epigenetic landscapes. In that case, one may have expected CDNs to be poorly shared among cancer types. However, as reported in Fig. 4 of the companion study, the sharing of CDNs across cancer types is far more common than the sharing of CDGs (Cancer Driving Genes). We suggest that CDNs have a much higher resolution than CDGs, whereby the signals are diluted by non-driver mutations. In other words, despite that the mutational landscape may be cancer-type specific, the pan-cancer selective pressure may be sufficiently high to permit the detection of CDN sharing among cancer types.

      Below, we shall respond in greater details. Epigenetic factors, such as chromatin states, methylation/acetylation levels, and replication timing, can provide valuable insights when analyzing mutational landscapes at a regional scale (Stamatoyannopoulos et al. 2009; Lawrence et al. 2013; Makova and Hardison 2015; Baylin and Jones 2016; Alexandrov et al. 2020; Abascal et al. 2021; Sherman et al. 2022). However, at the site-specific level, the effectiveness of these covariates in predicting mutational landscapes depends on their integration into a detailed model. Overemphasizing these covariates could lead to false negatives for known driver mutations (Hess et al. 2019; Elliott and Larsson 2021). In figure 3B of the main text, we illustrate the discrepancy between the mutation rate predictions from Dig and empirical observation. Ideally, no covariates would be needed under extensive sample sizes, where each mutable genomic sites would have sufficient mutations to yield a statistic significance and consequently, synonymous mutations would be sufficient for the characterization of mutational landscape. In this sense, the integration of mutational covariates represents a compromise under current sample size. In our study, the effect of unique mutational landscapes is captured by E(u), the mean mutation rate for each cancer type. We further accounted for the variability of site-level mutability using a gamma distribution. The primary goal of our study is to determine the upper limit of mutation recurrences under mutational mechanisms only. While selection force acts blindly to genomic features, mutational hotspots should exhibit common characteristics determined by their underlying mechanisms. In the main text, we attempted to identify such shared features among CDNs. Until these mutational mechanisms are fully understood, CDNs should be considered as potential driver mutations.

      (3) L223, the statement "In other words, the sequences surrounding the high-recurrence sites appear rather random.". Since it was a pan-cancer analysis, the unique patterns of each cancer type could be strongly diluted in the pan-cancer data.

      We now state that the analyses of mutation characteristic have been applied to the individual cancer types and did not find any pattern that deviates from randomness. Nevertheless, it may be argued that, with the exception of those with sufficiently large sample sizes such as lung and breast cancers, most datasets do not have the power to reject the null hypothesis. To alleviate this concern, we applied the ResNet and LSTM/GRU methods for the discovery of potential mutation motifs within each cancer type. All methods are more powerful than the one used but the results are the same – no cancer type yields a mutation pattern that can reject the null hypothesis of randomness (see below).

      As a positive control, we used these methods for the discovery of splicing sites of human exons. When aligned up with splicing site situated in the center (position 51 in the following plot), the sequence motif would look like:

      Author response image 1.

      5-prime

      Author response image 2.

      3-prime

      However, To account for the potential influence of distance from the mutant site in motif analysis, we randomly shuffled the splicing sites within a specified window around the alignment center, and their sequence logo now looks like:

      Author response image 3.

      5-prime shuffled

      Author response image 4.

      3-prime shuffled

      Author response image 5.

      random sequences from coding regions

      The classification results of the shuffled 5-prime (donner), 3-prime (acceptor) and random sequences from coding regions (Random CDS) are presented in the Author response table 1 (The accuracy for the aligned results, which is approximately 99%, is not shown here).

      Author response table 1.

      With the positive results from these positive controls (splicing site motifs) validating our methodology, we applied the same model structure to the train and test of potential mutational motifs of CDN sites. All models achieved approximately 50% accuracy in CDN motif analysis, suggesting that the sequence contexts surrounding CDN sites are not significantly different from other coding regions of the genome. This further implies that the recurrence of mutations at CDN sites is more likely driven by selection rather than mutational mechanisms.

      Note that this preliminary analysis may be limited by insufficient training data for CDN sites. Future studies will require larger sample sizes and more sophisticated models to address these limitations.

      (4) To solidify the findings, the results need to be replicated in an independent dataset.

      Figure 7 validates our CDN findings using the GENIE dataset, which primarily consists of targeted sequencing data from various panels. By focusing on the same genomic regions sequenced by GENIE, we observed a 3-5 fold increase in the number of discovered CDNs as sample size increased from approximately 1000 to 9000. Moreover, the majority of CDNs identified in TCGA were confirmed as CDNs in GENIE.

      (5) The key scripts and the list of key results (i.e., CDN sites with i{greater than or equal to}3) need to be shared to enable replication, validation, and further research. So far, only CDN sites with i{greater than or equal to}20 have been shared.

      We have now updated the “Data Availability” section in the main text, the corresponding scripts for key results are available on Gitlab at: https://gitlab.com/ultramicroevo/cdn_v1.

      (6) The versions of data used in this study are not clearly detailed, such as the specific version of gnomAD and the version and date of TCGA data downloaded from the GDC Data Portal.

      The versions of data sources have now been updated in the revised manuscript.

      Recommendations For The Authors:

      (1) L119, states "22.7 million nonsynonymous sites," but Table 1 lists the number as 22,540,623 (22.5 million). This discrepancy needs to be addressed for consistency.<br /> (2) Figure 2B, there is an unexplained drop in the line at i = 6 and 7 (from 83 to 45). Clarification is needed on why this drop occurs.<br /> (3) Figure 3A, for the CNS type, data for recurrence at 8 and 9 are missing. An explanation should be provided for this absence.<br /> (4) L201, the title refers to "100-mers," but L218 mentions "101-mers." This inconsistency needs to be corrected to ensure clarity and accuracy.<br /> (5) Figures 6 and 7 currently lack titles. Titles should be added to these figures to improve readability.

      Thanks. All corrections have been incorporated into the revised manuscript.

      Reviewer #2 (Public Review):<br /> Summary:<br /> The authors propose that cancer-driver mutations can be identified by Cancer Driving Nucleotides (CDNs). CDNs are defined as SNVs that occur frequently in genes. There are many ways to define cancer driver mutations, and the strengths and weaknesses are the reliance on statistics to define them.<br /> Strengths:<br /> There are many well-known approaches and studies that have already identified many canonical driver mutations. A potential strength is that mutation frequencies may be able to identify as yet unrecognized driver mutations. They use a previously developed method to estimate mutation hotspots across the genome (Dig, Sherman et al 2022). This publication has already used cancer sequence data to infer driver mutations based on higher-than-expected mutation frequencies. The advance here is to further illustrate that recurrent mutations (estimated at 3 or more mutations (CDNs) at the same base) are more likely to be the result of selection for a driver mutation (Figure 3). Further analysis indicates that mutation sequence context (Figure 4) or mutation mechanisms (Figure 5) are unlikely to be major causes for recurrent point mutations. Finally, they calculate (Figure 6) that most driver mutations identifiable by the CDN approach could be identified with about 100,000 to one million tumor coding genomes.<br /> Weaknesses:<br /> The manuscript does provide specific examples where recurrent mutations identify known driver mutations but do not identify "new" candidate driver mutations. Driver mutation validation is difficult and at least clinically, frequency (ie observed in multiple other cancer samples) is indeed commonly used to judge if an SNV has driver potential. The method would miss alternative ways to trigger driver alterations (translocations, indels, epigenetic, CNVs). Nevertheless, the value of the manuscript is its quantitative analysis of why mutation frequencies can identify cancer driver mutations.

      Recommendations For The Authors<br /> Whereas the analysis of driver mutations in WES has been extensive, the application of the method to WGS data (ie the noncoding regions) would provide new information.

      We appreciate that Reviewer #2 has suggested the potential application of our method to noncoding regions. Currently, the background mutation model is based on the site level mutations in coding regions, which hinders its direct applications in other mutation types such as CNVs, translocations and indels. We acknowledge that the proportion of patients with driver event involving CNV (73%) is comparable to that of coding point mutations (76%) as reported in the PCAWG analysis (Fig. 2A from Campbell et al., 2020). In future studies, we will attempt to establish a CNV-based background mutation rate model to identify positive selection signals driving tumorigenesis.

      References

      Abascal F, Harvey LMR, Mitchell E, Lawson ARJ, Lensing SV, Ellis P, Russell AJC, Alcantara RE, Baez-Ortega A, Wang Y, et al. 2021. Somatic mutation landscapes at single-molecule resolution. Nature:1–6.

      Alexandrov LB, Kim J, Haradhvala NJ, Huang MN, Tian Ng AW, Wu Y, Boot A, Covington KR, Gordenin DA, Bergstrom EN, et al. 2020. The repertoire of mutational signatures in human cancer. Nature 578:94–101.

      Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D, Weerasinghe A, Colaprico A, Wendl MC, Kim J, Reardon B, et al. 2018. Comprehensive Characterization of Cancer Driver Genes and Mutations. Cell 173:371-385.e18.

      Baylin SB, Jones PA. 2016. Epigenetic Determinants of Cancer. Cold Spring Harb Perspect Biol 8:a019505.

      Campbell PJ, Getz G, Korbel JO, Stuart JM, Jennings JL, Stein LD, Perry MD, Nahal-Bose HK, Ouellette BFF, Li CH, et al. 2020. Pan-cancer analysis of whole genomes. Nature 578:82–93.

      Drost J, Clevers H. 2018. Organoids in cancer research. Nat Rev Cancer 18:407–418.

      Elliott K, Larsson E. 2021. Non-coding driver mutations in human cancer. Nat Rev Cancer 21:500–509.

      Grzeskowiak CL, Kundu ST, Mo X, Ivanov AA, Zagorodna O, Lu H, Chapple RH, Tsang YH, Moreno D, Mosqueda M, et al. 2018. In vivo screening identifies GATAD2B as a metastasis driver in KRAS-driven lung cancer. Nat Commun 9:2732.

      Hess JM, Bernards A, Kim J, Miller M, Taylor-Weiner A, Haradhvala NJ, Lawrence MS, Getz G. 2019. Passenger Hotspot Mutations in Cancer. Cancer Cell 36:288-301.e14.

      Hodis E, Triglia ET, Kwon JYH, Biancalani T, Zakka LR, Parkar S, Hütter J-C, Buffoni L, Delorey TM, Phillips D, et al. 2022. Stepwise-edited, human melanoma models reveal mutations’ effect on tumor and microenvironment. Science 376:eabi8175.

      Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL, Stewart C, Mermel CH, Roberts SA, et al. 2013. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499:214–218.

      Makova KD, Hardison RC. 2015. The effects of chromatin organization on variation in mutation rates in the genome. Nat Rev Genet 16:213–223.

      Michels BE, Mosa MH, Streibl BI, Zhan T, Menche C, Abou-El-Ardat K, Darvishi T, Członka E, Wagner S, Winter J, et al. 2020. Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids. Cell Stem Cell 26:782-792.e7.

      Ng PK-S, Li J, Jeong KJ, Shao S, Chen H, Tsang YH, Sengupta S, Wang Z, Bhavana VH, Tran R, et al. 2018. Systematic Functional Annotation of Somatic Mutations in Cancer. Cancer Cell 33:450-462.e10.

      Sherman MA, Yaari AU, Priebe O, Dietlein F, Loh P-R, Berger B. 2022. Genome-wide mapping of somatic mutation rates uncovers drivers of cancer. Nat Biotechnol 40:1634–1643.

      Stamatoyannopoulos JA, Adzhubei I, Thurman RE, Kryukov GV, Mirkin SM, Sunyaev SR. 2009. Human mutation rate associated with DNA replication timing. Nat Genet 41:393–395.

  2. Aug 2024
    1. The Gilgamesh Epic is the most notable literary product of Babylonia as yet discovered in the mounds of Mesopotamia. It recounts the exploits and adventures of a favorite hero, and in its final form covers twelve tablets, each tablet consisting of six columns (three on the obverse and three on the reverse) of about 50 lines for each column, or a total of about 3600 lines. Of this total, however, barely more than one-half has been found among the remains of the great collection of cuneiform tablets gathered by King Ashurbanapal (668–626 B.C.) in his palace at Nineveh, and discovered by Layard in 18541 in the course of his excavations of the mound Kouyunjik (opposite Mosul). The fragments of the epic painfully gathered—chiefly by George Smith—from the circa 30,000 tablets and bits of tablets brought to the British Museum were published in model form by Professor Paul Haupt;2 and that edition still remains the primary source for our study of the Epic. [10] For the sake of convenience we may call the form of the Epic in the fragments from the library of Ashurbanapal the Assyrian version, though like most of the literary productions in the library it not only reverts to a Babylonian original, but represents a late copy of a much older original. The absence of any reference to Assyria in the fragments recovered justifies us in assuming that the Assyrian version received its present form in Babylonia, perhaps in Erech; though it is of course possible that some of the late features, particularly the elaboration of the teachings of the theologians or schoolmen in the eleventh and twelfth tablets, may have been produced at least in part under Assyrian influence. A definite indication that the Gilgamesh Epic reverts to a period earlier than Hammurabi (or Hammurawi)3 i.e., beyond 2000 B. C., was furnished by the publication of a text clearly belonging to the first Babylonian dynasty (of which Hammurabi was the sixth member) in CT. VI, 5; which text Zimmern4 recognized as a part of the tale of Atra-ḫasis, one of the names given to the survivor of the deluge, recounted on the eleventh tablet of the Gilgamesh Epic.5 This was confirmed by the discovery6 of a [11]fragment of the deluge story dated in the eleventh year of Ammisaduka, i.e., c. 1967 B.C. In this text, likewise, the name of the deluge hero appears as Atra-ḫasis (col. VIII, 4).7 But while these two tablets do not belong to the Gilgamesh Epic and merely introduce an episode which has also been incorporated into the Epic, Dr. Bruno Meissner in 1902 published a tablet, dating, as the writing and the internal evidence showed, from the Hammurabi period, which undoubtedly is a portion of what by way of distinction we may call an old Babylonian version.8 It was picked up by Dr. Meissner at a dealer’s shop in Bagdad and acquired for the Berlin Museum. The tablet consists of four columns (two on the obverse and two on the reverse) and deals with the hero’s wanderings in search of a cure from disease with which he has been smitten after the death of his companion Enkidu. The hero fears that the disease will be fatal and longs to escape death. It corresponds to a portion of Tablet X of the Assyrian version. Unfortunately, only the lower portion of the obverse and the upper of the reverse have been preserved (57 lines in all); and in default of a colophon we do not know the numeration of the tablet in this old Babylonian edition. Its chief value, apart from its furnishing a proof for the existence of the Epic as early as 2000 B. C., lies (a) in the writing Gish instead of Gish-gi(n)-mash in the Assyrian version, for the name of the hero, (b) in the writing En-ki-dũ—abbreviated from dũg—() “Enki is good” for En-ki-dú () in the Assyrian version,9 and (c) in the remarkable address of the maiden Sabitum, dwelling at the seaside, to whom Gilgamesh comes in the course of his wanderings. From the Assyrian version we know that the hero tells the maiden of his grief for his lost companion, and of his longing to escape the dire fate of Enkidu. In the old Babylonian fragment the answer of Sabitum is given in full, and the sad note that it strikes, showing how hopeless it is for man to try to escape death which is in store for all mankind, is as remarkable as is the philosophy of “eat, drink and be merry” which Sabitum imparts. The address indicates how early the tendency arose to attach to ancient tales the current religious teachings. [12] “Why, O Gish, does thou run about? The life that thou seekest, thou wilt not find. When the gods created mankind, Death they imposed on mankind; Life they kept in their power. Thou, O Gish, fill thy belly, Day and night do thou rejoice, Daily make a rejoicing! Day and night a renewal of jollification! Let thy clothes be clean, Wash thy head and pour water over thee! Care for the little one who takes hold of thy hand! Let the wife rejoice in thy bosom!” Such teachings, reminding us of the leading thought in the Biblical Book of Ecclesiastes,10 indicate the didactic character given to ancient tales that were of popular origin, but which were modified and elaborated under the influence of the schools which arose in connection with the Babylonian temples. The story itself belongs, therefore, to a still earlier period than the form it received in this old Babylonian version. The existence of this tendency at so early a date comes to us as a genuine surprise, and justifies the assumption that the attachment of a lesson to the deluge story in the Assyrian version, to wit, the limitation in attainment of immortality to those singled out by the gods as exceptions, dates likewise from the old Babylonian period. The same would apply to the twelfth tablet, which is almost entirely didactic, intended to illustrate the impossibility of learning anything of the fate of those who have passed out of this world. It also emphasizes the necessity of contenting oneself with the comfort that the care of the dead, by providing burial and food and drink offerings for them affords, as the only means of ensuring for them rest and freedom from the pangs of hunger and distress. However, it is of course possible that the twelfth tablet, which impresses one as a supplement to the adventures of Gilgamesh, ending with his return to Uruk (i.e., Erech) at the close of the eleventh tablet, may represent a later elaboration of the tendency to connect religious teachings with the exploits of a favorite hero. [13] We now have further evidence both of the extreme antiquity of the literary form of the Gilgamesh Epic and also of the disposition to make the Epic the medium of illustrating aspects of life and the destiny of mankind. The discovery by Dr. Arno Poebel of a Sumerian form of the tale of the descent of Ishtar to the lower world and her release11—apparently a nature myth to illustrate the change of season from summer to winter and back again to spring—enables us to pass beyond the Akkadian (or Semitic) form of tales current in the Euphrates Valley to the Sumerian form. Furthermore, we are indebted to Dr. Langdon for the identification of two Sumerian fragments in the Nippur Collection which deal with the adventures of Gilgamesh, one in Constantinople,12 the other in the collection of the University of Pennsylvania Museum.13 The former, of which only 25 lines are preserved (19 on the obverse and 6 on the reverse), appears to be a description of the weapons of Gilgamesh with which he arms himself for an encounter—presumably the encounter with Ḫumbaba or Ḫuwawa, the ruler of the cedar forest in the mountain.14 The latter deals with the building operations of Gilgamesh in the city of Erech. A text in Zimmern’s Sumerische Kultlieder aus altbabylonischer Zeit (Leipzig, 1913), No. 196, appears likewise to be a fragment of the Sumerian version of the Gilgamesh Epic, bearing on the episode of Gilgamesh’s and Enkidu’s relations to the goddess Ishtar, covered in the sixth and seventh tablets of the Assyrian version.15 Until, however, further fragments shall have turned up, it would be hazardous to institute a comparison between the Sumerian and the Akkadian versions. All that can be said for the present is that there is every reason to believe in the existence of a literary form of the Epic in Sumerian which presumably antedated the Akkadian recension, [14]just as we have a Sumerian form of Ishtar’s descent into the nether world, and Sumerian versions of creation myths, as also of the Deluge tale.16 It does not follow, however, that the Akkadian versions of the Gilgamesh Epic are translations of the Sumerian, any more than that the Akkadian creation myths are translations of a Sumerian original. Indeed, in the case of the creation myths, the striking difference between the Sumerian and Akkadian views of creation17 points to the independent production of creation stories on the part of the Semitic settlers of the Euphrates Valley, though no doubt these were worked out in part under Sumerian literary influences. The same is probably true of Deluge tales, which would be given a distinctly Akkadian coloring in being reproduced and steadily elaborated by the Babylonian literati attached to the temples. The presumption is, therefore, in favor of an independent literary origin for the Semitic versions of the Gilgamesh Epic, though naturally with a duplication of the episodes, or at least of some of them, in the Sumerian narrative. Nor does the existence of a Sumerian form of the Epic necessarily prove that it originated with the Sumerians in their earliest home before they came to the Euphrates Valley. They may have adopted it after their conquest of southern Babylonia from the Semites who, there are now substantial grounds for believing, were the earlier settlers in the Euphrates Valley.18 We must distinguish, therefore, between the earliest literary form, which was undoubtedly Sumerian, and the origin of the episodes embodied in the Epic, including the chief actors, Gilgamesh and his companion Enkidu. It will be shown that one of the chief episodes, the encounter of the two heroes with a powerful guardian or ruler of a cedar forest, points to a western region, more specifically to Amurru, as the scene. The names of the two chief actors, moreover, appear to have been “Sumerianized” by an artificial process,19 and if this view turns out to be [15]correct, we would have a further ground for assuming the tale to have originated among the Akkadian settlers and to have been taken over from them by the Sumerians. New light on the earliest Babylonian version of the Epic, as well as on the Assyrian version, has been shed by the recovery of two substantial fragments of the form which the Epic had assumed in Babylonia in the Hammurabi period. The study of this important new material also enables us to advance the interpretation of the Epic and to perfect the analysis into its component parts. In the spring of 1914, the Museum of the University of Pennsylvania acquired by purchase a large tablet, the writing of which as well as the style and the manner of spelling verbal forms and substantives pointed distinctly to the time of the first Babylonian dynasty. The tablet was identified by Dr. Arno Poebel as part of the Gilgamesh Epic; and, as the colophon showed, it formed the second tablet of the series. He copied it with a view to publication, but the outbreak of the war which found him in Germany—his native country—prevented him from carrying out this intention.20 He, however, utilized some of its contents in his discussion of the historical or semi-historical traditions about Gilgamesh, as revealed by the important list of partly mythical and partly historical dynasties, found among the tablets of the Nippur collection, in which Gilgamesh occurs21 as a King of an Erech dynasty, whose father was Â, a priest of Kulab.22 The publication of the tablet was then undertaken by Dr. Stephen Langdon in monograph form under the title, “The Epic of Gilgamish.”23 In a preliminary article on the tablet in the Museum Journal, Vol. VIII, pages 29–38, Dr. Langdon took the tablet to be of the late [16]Persian period (i.e., between the sixth and third century B. C.), but his attention having been called to this error of some 1500 years, he corrected it in his introduction to his edition of the text, though he neglected to change some of his notes in which he still refers to the text as “late.”24 In addition to a copy of the text, accompanied by a good photograph, Dr. Langdon furnished a transliteration and translation with some notes and a brief introduction. The text is unfortunately badly copied, being full of errors; and the translation is likewise very defective. A careful collation with the original tablet was made with the assistance of Dr. Edward Chiera, and as a consequence we are in a position to offer to scholars a correct text. We beg to acknowledge our obligations to Dr. Gordon, the Director of the Museum of the University of Pennsylvania, for kindly placing the tablet at our disposal. Instead of republishing the text, I content myself with giving a full list of corrections in the appendix to this volume which will enable scholars to control our readings, and which will, I believe, justify the translation in the numerous passages in which it deviates from Dr. Langdon’s rendering. While credit should be given to Dr. Langdon for having made this important tablet accessible, the interests of science demand that attention be called to his failure to grasp the many important data furnished by the tablet, which escaped him because of his erroneous readings and faulty translations. The tablet, consisting of six columns (three on the obverse and three on the reverse), comprised, according to the colophon, 240 lines25 and formed the second tablet of the series. Of the total, 204 lines are preserved in full or in part, and of the missing thirty-six quite a number can be restored, so that we have a fairly complete tablet. The most serious break occurs at the top of the reverse, where about eight lines are missing. In consequence of this the connection between the end of the obverse (where about five lines are missing) and the beginning of the reverse is obscured, though not to the extent of our entirely losing the thread of the narrative. [17] About the same time that the University of Pennsylvania Museum purchased this second tablet of the Gilgamesh Series, Yale University obtained a tablet from the same dealer, which turned out to be a continuation of the University of Pennsylvania tablet. That the two belong to the same edition of the Epic is shown by their agreement in the dark brown color of the clay, in the writing as well as in the size of the tablet, though the characters on the Yale tablet are somewhat cramped and in consequence more difficult to read. Both tablets consist of six columns, three on the obverse and three on the reverse. The measurements of both are about the same, the Pennsylvania tablet being estimated at about 7 inches high, as against 72/16 inches for the Yale tablet, while the width of both is 6½ inches. The Yale tablet is, however, more closely written and therefore has a larger number of lines than the Pennsylvania tablet. The colophon to the Yale tablet is unfortunately missing, but from internal evidence it is quite certain that the Yale tablet follows immediately upon the Pennsylvania tablet and, therefore, may be set down as the third of the series. The obverse is very badly preserved, so that only a general view of its contents can be secured. The reverse contains serious gaps in the first and second columns. The scribe evidently had a copy before him which he tried to follow exactly, but finding that he could not get all of the copy before him in the six columns, he continued the last column on the edge. In this way we obtain for the sixth column 64 lines as against 45 for column IV, and 47 for column V, and a total of 292 lines for the six columns. Subtracting the 16 lines written on the edge leaves us 276 lines for our tablet as against 240 for its companion. The width of each column being the same on both tablets, the difference of 36 lines is made up by the closer writing. Both tablets have peculiar knobs at the sides, the purpose of which is evidently not to facilitate holding the tablet in one’s hand while writing or reading it, as Langdon assumed26 (it would be quite impracticable for this purpose), but simply to protect the tablet in its position on a shelf, where it would naturally be placed on the edge, just as we arrange books on a shelf. Finally be it noted that these two tablets of the old Babylonian version do not belong to the same edition as the Meissner tablet above described, for the latter consists [18]of two columns each on obverse and reverse, as against three columns each in the case of our two tablets. We thus have the interesting proof that as early as 2000 B.C. there were already several editions of the Epic. As to the provenance of our two tablets, there are no definite data, but it is likely that they were found by natives in the mounds at Warka, from which about the year 1913, many tablets came into the hands of dealers. It is likely that where two tablets of a series were found, others of the series were also dug up, and we may expect to find some further portions of this old Babylonian version turning up in the hands of other dealers or in museums. Coming to the contents of the two tablets, the Pennsylvania tablet deals with the meeting of the two heroes, Gilgamesh and Enkidu, their conflict, followed by their reconciliation, while the Yale tablet in continuation takes up the preparations for the encounter of the two heroes with the guardian of the cedar forest, Ḫumbaba—but probably pronounced Ḫubaba27—or, as the name appears in the old Babylonian version, Ḫuwawa. The two tablets correspond, therefore, to portions of Tablets I to V of the Assyrian version;28 but, as will be shown in detail further on, the number of completely parallel passages is not large, and the Assyrian version shows an independence of the old Babylonian version that is larger than we had reason to expect. In general, it may be said that the Assyrian version is more elaborate, which points to its having received its present form at a considerably later period than the old Babylonian version.29 On the other hand, we already find in the Babylonian version the tendency towards repetition, which is characteristic of Babylonian-Assyrian tales in general. Through the two Babylonian tablets we are enabled to fill out certain details [19]of the two episodes with which they deal: (1) the meeting of Gilgamesh and Enkidu, and (2) the encounter with Ḫuwawa; while their greatest value consists in the light that they throw on the gradual growth of the Epic until it reached its definite form in the text represented by the fragments in Ashurbanapal’s Library. Let us now take up the detailed analysis, first of the Pennsylvania tablet and then of the Yale tablet. The Pennsylvania tablet begins with two dreams recounted by Gilgamesh to his mother, which the latter interprets as presaging the coming of Enkidu to Erech. In the one, something like a heavy meteor falls from heaven upon Gilgamesh and almost crushes him. With the help of the heroes of Erech, Gilgamesh carries the heavy burden to his mother Ninsun. The burden, his mother explains, symbolizes some one who, like Gilgamesh, is born in the mountains, to whom all will pay homage and of whom Gilgamesh will become enamoured with a love as strong as that for a woman. In a second dream, Gilgamesh sees some one who is like him, who brandishes an axe, and with whom he falls in love. This personage, the mother explains, is again Enkidu. Langdon is of the opinion that these dreams are recounted to Enkidu by a woman with whom Enkidu cohabits for six days and seven nights and who weans Enkidu from association with animals. This, however, cannot be correct. The scene between Enkidu and the woman must have been recounted in detail in the first tablet, as in the Assyrian version,30 whereas here in the second tablet we have the continuation of the tale with Gilgamesh recounting his dreams directly to his mother. The story then continues with the description of the coming of Enkidu, conducted by the woman to the outskirts of Erech, where food is given him. The main feature of the incident is the conversion of Enkidu to civilized life. Enkidu, who hitherto had gone about naked, is clothed by the woman. Instead of sucking milk and drinking from a trough like an animal, food and strong drink are placed before him, and he is taught how to eat and drink in human fashion. In human fashion he also becomes drunk, and his “spree” is naïvely described: “His heart became glad and his face shone.”31 [20]Like an animal, Enkidu’s body had hitherto been covered with hair, which is now shaved off. He is anointed with oil, and clothed “like a man.” Enkidu becomes a shepherd, protecting the fold against wild beasts, and his exploit in dispatching lions is briefly told. At this point—the end of column 3 (on the obverse), i.e., line 117, and the beginning of column 4 (on the reverse), i.e., line 131—a gap of 13 lines—the tablet is obscure, but apparently the story of Enkidu’s gradual transformation from savagery to civilized life is continued, with stress upon his introduction to domestic ways with the wife chosen or decreed for him, and with work as part of his fate. All this has no connection with Gilgamesh, and it is evident that the tale of Enkidu was originally an independent tale to illustrate the evolution of man’s career and destiny, how through intercourse with a woman he awakens to the sense of human dignity, how he becomes accustomed to the ways of civilization, how he passes through the pastoral stage to higher walks of life, how the family is instituted, and how men come to be engaged in the labors associated with human activities. In order to connect this tale with the Gilgamesh story, the two heroes are brought together; the woman taking on herself, in addition to the rôle of civilizer, that of the medium through which Enkidu is brought to Gilgamesh. The woman leads Enkidu from the outskirts of Erech into the city itself, where the people on seeing him remark upon his likeness to Gilgamesh. He is the very counterpart of the latter, though somewhat smaller in stature. There follows the encounter between the two heroes in the streets of Erech, where they engage in a fierce combat. Gilgamesh is overcome by Enkidu and is enraged at being thrown to the ground. The tablet closes with the endeavor of Enkidu to pacify Gilgamesh. Enkidu declares that the mother of Gilgamesh has exalted her son above the ordinary mortal, and that Enlil himself has singled him out for royal prerogatives. After this, we may assume, the two heroes become friends and together proceed to carry out certain exploits, the first of which is an attack upon the mighty guardian of the cedar forest. This is the main episode in the Yale tablet, which, therefore, forms the third tablet of the old Babylonian version. In the first column of the obverse of the Yale tablet, which is badly preserved, it would appear that the elders of Erech (or perhaps the people) are endeavoring to dissuade Gilgamesh from making the [21]attempt to penetrate to the abode of Ḫuwawa. If this is correct, then the close of the first column may represent a conversation between these elders and the woman who accompanies Enkidu. It would be the elders who are represented as “reporting the speech to the woman,” which is presumably the determination of Gilgamesh to fight Ḫuwawa. The elders apparently desire Enkidu to accompany Gilgamesh in this perilous adventure, and with this in view appeal to the woman. In the second column after an obscure reference to the mother of Gilgamesh—perhaps appealing to the sun-god—we find Gilgamesh and Enkidu again face to face. From the reference to Enkidu’s eyes “filled with tears,” we may conclude that he is moved to pity at the thought of what will happen to Gilgamesh if he insists upon carrying out his purpose. Enkidu, also, tries to dissuade Gilgamesh. This appears to be the main purport of the dialogue between the two, which begins about the middle of the second column and extends to the end of the third column. Enkidu pleads that even his strength is insufficient, “My arms are lame, My strength has become weak.” (lines 88–89) Gilgamesh apparently asks for a description of the terrible tyrant who thus arouses the fear of Enkidu, and in reply Enkidu tells him how at one time, when he was roaming about with the cattle, he penetrated into the forest and heard the roar of Ḫuwawa which was like that of a deluge. The mouth of the tyrant emitted fire, and his breath was death. It is clear, as Professor Haupt has suggested,32 that Enkidu furnishes the description of a volcano in eruption, with its mighty roar, spitting forth fire and belching out a suffocating smoke. Gilgamesh is, however, undaunted and urges Enkidu to accompany him in the adventure. “I will go down to the forest,” says Gilgamesh, if the conjectural restoration of the line in question (l. 126) is correct. Enkidu replies by again drawing a lurid picture of what will happen “When we go (together) to the forest…….” This speech of Enkidu is continued on the reverse. In reply Gilgamesh emphasizes his reliance upon the good will of Shamash and reproaches Enkidu with cowardice. He declares himself superior to Enkidu’s warning, and in bold terms [22]says that he prefers to perish in the attempt to overcome Ḫuwawa rather than abandon it. “Wherever terror is to be faced, Thou, forsooth, art in fear of death. Thy prowess lacks strength. I will go before thee, Though thy mouth shouts to me: ‘thou art afraid to approach,’ If I fall, I will establish my name.” (lines 143–148) There follows an interesting description of the forging of the weapons for the two heroes in preparation for the encounter.33 The elders of Erech when they see these preparations are stricken with fear. They learn of Ḫuwawa’s threat to annihilate Gilgamesh if he dares to enter the cedar forest, and once more try to dissuade Gilgamesh from the undertaking. “Thou art young, O Gish, and thy heart carries thee away, Thou dost not know what thou proposest to do.” (lines 190–191) They try to frighten Gilgamesh by repeating the description of the terrible Ḫuwawa. Gilgamesh is still undaunted and prays to his patron deity Shamash, who apparently accords him a favorable “oracle” (têrtu). The two heroes arm themselves for the fray, and the elders of Erech, now reconciled to the perilous undertaking, counsel Gilgamesh to take provision along for the undertaking. They urge Gilgamesh to allow Enkidu to take the lead, for “He is acquainted with the way, he has trodden the road [to] the entrance of the forest.” (lines 252–253) The elders dismiss Gilgamesh with fervent wishes that Enkidu may track out the “closed path” for Gilgamesh, and commit him to the care of Lugalbanda—here perhaps an epithet of Shamash. They advise Gilgamesh to perform certain rites, to wash his feet in the stream of Ḫuwawa and to pour out a libation of water to Shamash. Enkidu follows in a speech likewise intended to encourage the hero; and with the actual beginning of the expedition against Ḫuwawa the tablet ends. The encounter itself, with the triumph of the two heroes, must have been described in the fourth tablet. [23] Now before taking up the significance of the additions to our knowledge of the Epic gained through these two tablets, it will be well to discuss the forms in which the names of the two heroes and of the ruler of the cedar forest occur in our tablets. As in the Meissner fragment, the chief hero is invariably designated as dGish in both the Pennsylvania and Yale tablets; and we may therefore conclude that this was the common form in the Hammurabi period, as against the writing dGish-gì(n)-mash34 in the Assyrian version. Similarly, as in the Meissner fragment, the second hero’s name is always written En-ki-dũ35 (abbreviated from dúg) as against En-ki-dú in the Assyrian version. Finally, we encounter in the Yale tablet for the first time the writing Ḫu-wa-wa as the name of the guardian of the cedar forest, as against Ḫum-ba-ba in the Assyrian version, though in the latter case, as we may now conclude from the Yale tablet, the name should rather be read Ḫu-ba-ba.36 The variation in the writing of the latter name is interesting as pointing to the aspirate pronunciation of the labial in both instances. The name would thus present a complete parallel to the Hebrew name Ḫowawa (or Ḫobab) who appears as the brother-in-law of Moses in the P document, Numbers 10, 29.37 Since the name also occurs, written precisely as in the Yale tablet, among the “Amoritic” names in the important lists published by Dr. Chiera,38 there can be no doubt that [24]Ḫuwawa or Ḫubaba is a West Semitic name. This important fact adds to the probability that the “cedar forest” in which Ḫuwawa dwells is none other than the Lebanon district, famed since early antiquity for its cedars. This explanation of the name Ḫuwawa disposes of suppositions hitherto brought forward for an Elamitic origin. Gressmann39 still favors such an origin, though realizing that the description of the cedar forest points to the Amanus or Lebanon range. In further confirmation of the West Semitic origin of the name, we have in Lucian, De Dea Syria, § 19, the name Kombabos40 (the guardian of Stratonika), which forms a perfect parallel to Ḫu(m)baba. Of the important bearings of this western character of the name Ḫuwawa on the interpretation and origin of the Gilgamesh Epic, suggesting that the episode of the encounter between the tyrant and the two heroes rests upon a tradition of an expedition against the West or Amurru land, we shall have more to say further on. The variation in the writing of the name Enkidu is likewise interesting. It is evident that the form in the old Babylonian version with the sign dũ (i.e., dúg) is the original, for it furnishes us with a suitable etymology “Enki is good.” The writing with dúg, pronounced dū, also shows that the sign dú as the third element in the form which the name has in the Assyrian version is to be read dú, and that former readings like Ea-bani must be definitely abandoned.41 The form with dú is clearly a phonetic writing of the Sumerian name, the sign dú being chosen to indicate the pronunciation (not the ideograph) of the third element dúg. This is confirmed by the writing En-gi-dú in the syllabary CT XVIII, 30, 10. The phonetic writing is, therefore, a warning against any endeavor to read the name by an Akkadian transliteration of the signs. This would not of itself prove that Enkidu is of Sumerian origin, for it might well be that the writing En-ki-dú is an endeavor to give a Sumerian aspect to a name that may have been foreign. The element dúg corresponds to the Semitic ṭâbu, “good,” and En-ki being originally a designation of a deity as the “lord of the land,” which would be the Sumerian [25]manner of indicating a Semitic Baal, it is not at all impossible that En-ki-dúg may be the “Sumerianized” form of a Semitic בַּעל טזֹב “Baal is good.” It will be recalled that in the third column of the Yale tablet, Enkidu speaks of himself in his earlier period while still living with cattle, as wandering into the cedar forest of Ḫuwawa, while in another passage (ll. 252–253) he is described as “acquainted with the way … to the entrance of the forest.” This would clearly point to the West as the original home of Enkidu. We are thus led once more to Amurru—taken as a general designation of the West—as playing an important role in the Gilgamesh Epic.42 If Gilgamesh’s expedition against Ḫuwawa of the Lebanon district recalls a Babylonian campaign against Amurru, Enkidu’s coming from his home, where, as we read repeatedly in the Assyrian version, “He ate herbs with the gazelles, Drank out of a trough with cattle,”43 may rest on a tradition of an Amorite invasion of Babylonia. The fight between Gilgamesh and Enkidu would fit in with this tradition, while the subsequent reconciliation would be the form in which the tradition would represent the enforced union between the invaders and the older settlers. Leaving this aside for the present, let us proceed to a consideration of the relationship of the form dGish, for the chief personage in the Epic in the old Babylonian version, to dGish-gi(n)-mash in the Assyrian version. Of the meaning of Gish there is fortunately no doubt. It is clearly the equivalent to the Akkadian zikaru, “man” (Brünnow No. 5707), or possibly rabû, “great” (Brünnow No. 5704). Among various equivalents, the preference is to be given to itlu, “hero.” The determinative for deity stamps the person so designated as deified, or as in part divine, and this is in accord with the express statement in the Assyrian version of the Gilgamesh Epic which describes the hero as “Two-thirds god and one-third human.”44 [26]Gish is, therefore, the hero-god par excellence; and this shows that we are not dealing with a genuine proper name, but rather with a descriptive attribute. Proper names are not formed in this way, either in Sumerian or Akkadian. Now what relation does this form Gish bear to as the name of the hero is invariably written in the Assyrian version, the form which was at first read dIz-tu-bar or dGish-du-bar by scholars, until Pinches found in a neo-Babylonian syllabary45 the equation of it with Gi-il-ga-mesh? Pinches’ discovery pointed conclusively to the popular pronunciation of the hero’s name as Gilgamesh; and since Aelian (De natura Animalium XII, 2) mentions a Babylonian personage Gilgamos (though what he tells us of Gilgamos does not appear in our Epic, but seems to apply to Etana, another figure of Babylonian mythology), there seemed to be no further reason to question that the problem had been solved. Besides, in a later Syriac list of Babylonian kings found in the Scholia of Theodor bar Koni, the name גלמגום with a variant גמיגמוס occurs,46 and it is evident that we have here again the Gi-il-ga-mesh, discovered by Pinches. The existence of an old Babylonian hero Gilgamesh who was likewise a king is thus established, as well as his identification with It is evident that we cannot read this name as Iz-tu-bar or Gish-du-bar, but that we must read the first sign as Gish and the third as Mash, while for the second we must assume a reading Gìn or Gi. This would give us Gish-gì(n)-mash which is clearly again (like En-ki-dú) not an etymological writing but a phonetic one, intended to convey an approach to the popular pronunciation. Gi-il-ga-mesh might well be merely a variant for Gish-ga-mesh, or vice versa, and this would come close to Gish-gi-mash. Now, when we have a name the pronunciation of which is not definite but approximate, and which is written in various ways, the probabilities are that the name is foreign. A foreign name might naturally be spelled in various ways. The [27]Epic in the Assyrian version clearly depicts dGish-gì(n)-mash as a conqueror of Erech, who forces the people into subjection, and whose autocratic rule leads the people of Erech to implore the goddess Aruru to create a rival to him who may withstand him. In response to this appeal dEnkidu is formed out of dust by Aruru and eventually brought to Erech.47 Gish-gì(n)-mash or Gilgamesh is therefore in all probability a foreigner; and the simplest solution suggested by the existence of the two forms (1) Gish in the old Babylonian version and (2) Gish-gì(n)-mash in the Assyrian version, is to regard the former as an abbreviation, which seemed appropriate, because the short name conveyed the idea of the “hero” par excellence. If Gish-gì(n)-mash is a foreign name, one would think in the first instance of Sumerian; but here we encounter a difficulty in the circumstance that outside of the Epic this conqueror and ruler of Erech appears in quite a different form, namely, as dGish-bil-ga-mesh, with dGish-gibil(or bìl)-ga-mesh and dGish-bil-ge-mesh as variants.48 In the remarkable list of partly mythological and partly historical dynasties, published by Poebel,49 the fifth member of the first dynasty of Erech appears as dGish-bil-ga-mesh; and similarly in an inscription of the days of Sin-gamil, dGish-bil-ga-mesh is mentioned as the builder of the wall of Erech.50 Moreover, in the several fragments of the Sumerian version of the Epic we have invariably the form dGish-bil-ga-mesh. It is evident, therefore, that this is the genuine form of the name in Sumerian and presumably, therefore, the oldest form. By way of further confirmation we have in the syllabary above referred to, CT, XVIII, 30, 6–8, three designations of our hero, viz: dGish-gibil(or bíl)-ga-mesh muḳ-tab-lu (“warrior”) a-lik pa-na (“leader”) All three designations are set down as the equivalent of the Sumerian Esigga imin i.e., “the seven-fold hero.” [28] Of the same general character is the equation in another syllabary:51 Esigga-tuk and its equivalent Gish-tuk = “the one who is a hero.” Furthermore, the name occurs frequently in “Temple” documents of the Ur dynasty in the form dGish-bil-ga-mesh52 with dGish-bil-gi(n)-mesh as a variant.53 In a list of deities (CT XXV, 28, K 7659) we likewise encounter dGish-gibil(or bíl)-ga-mesh, and lastly in a syllabary we have the equation54 dGish-gi-mas-[si?] = dGish-bil-[ga-mesh]. The variant Gish-gibil for Gish-bil may be disposed of readily, in view of the frequent confusion or interchange of the two signs Bil (Brünnow No. 4566) and Gibil or Bíl (Brünnow No. 4642) which has also the value Gi (Brünnow 4641), so that we might also read Gish-gi-ga-mesh. Both signs convey the idea of “fire,” “renew,” etc.; both revert to the picture of flames of fire, in the one case with a bowl (or some such obiect) above it, in the other the flames issuing apparently from a torch.55 The meaning of the name is not affected whether we read dGish-bil-ga-mesh or dGish-gibil(or bíl)-ga-mesh, for the middle element in the latter case being identical with the fire-god, written dBil-gi and to be pronounced in the inverted form as Gibil with -ga (or ge) as the phonetic complement; it is equivalent, therefore, to the writing bil-ga in the former case. Now Gish-gibil or Gish-bíl conveys the idea of abu, “father” (Brünnow No. 5713), just as Bil (Brünnow No. 4579) has this meaning, while Pa-gibil-(ga) or Pa-bíl-ga is abu abi, “grandfather.”56 This meaning may be derived from Gibil, as also from Bíl = išatu, “fire,” then eššu, “new,” then abu, “father,” as the renewer or creator. Gish with Bíl or Gibil would, therefore, be “the father-man” or “the father-hero,” [29]i.e., again the hero par excellence, the original hero, just as in Hebrew and Arabic ab is used in this way.57 The syllable ga being a phonetic complement, the element mesh is to be taken by itself and to be explained, as Poebel suggested, as “hero” (itlu. Brünnow No. 5967). We would thus obtain an entirely artificial combination, “man (or hero), father, hero,” which would simply convey in an emphatic manner the idea of the Ur-held, the original hero, the father of heroes as it were—practically the same idea, therefore, as the one conveyed by Gish alone, as the hero par excellence. Our investigation thus leads us to a substantial identity between Gish and the longer form Gish-bil(or bíl)-ga-mesh, and the former might, therefore, well be used as an abbreviation of the latter. Both the shorter and the longer forms are descriptive epithets based on naive folk etymology, rather than personal names, just as in the designation of our hero as muḳtablu, the “fighter,” or as âlik pâna, “the leader,” or as Esigga imin, “the seven-fold hero,” or Esigga tuk, “the one who is a hero,” are descriptive epithets, and as Atra-ḫasis, “the very wise one,” is such an epithet for the hero of the deluge story. The case is different with Gi-il-ga-mesh, or Gish-gì(n)-mash, which represent the popular and actual pronunciation of the name, or at least the approach to such pronunciation. Such forms, stripped as they are of all artificiality, impress one as genuine names. The conclusion to which we are thus led is that Gish-bil(or bíl)-ga-mesh is a play upon the genuine name, to convey to those to whom the real name, as that of a foreigner, would suggest no meaning an interpretation fitting in with his character. In other words, Gish-bil-ga-mesh is a “Sumerianized” form of the name, introduced into the Sumerian version of the tale which became a folk-possession in the Euphrates Valley. Such plays upon names to suggest the character of an individual or some incident are familiar to us from the narratives in Genesis.58 They do not constitute genuine etymologies and are rarely of use in leading to a correct etymology. Reuben, e.g., certainly does not mean “Yahweh has seen my affliction,” which the mother is supposed to have exclaimed at [30]the birth (Genesis 29, 32), with a play upon ben and be’onyi, any more than Judah means “I praise Yahweh” (v. 35), though it does contain the divine name (Yehô) as an element. The play on the name may be close or remote, as long as it fulfills its function of suggesting an etymology that is complimentary or appropriate. In this way, an artificial division and at the same time a distortion of a foreign name like Gilgamesh into several elements, Gish-bil-ga-mesh, is no more violent than, for example, the explanation of Issachar or rather Issaschar as “God has given my hire” (Genesis 30, 18) with a play upon the element sechar, and as though the name were to be divided into Yah (“God”) and sechar (“hire”); or the popular name of Alexander among the Arabs as Zu’l Karnaini, “the possessor of the two horns.” with a suggestion of his conquest of two hemispheres, or what not.59 The element Gil in Gilgamesh would be regarded as a contraction of Gish-bil or gi-bil, in order to furnish the meaning “father-hero,” or Gil might be looked upon as a variant for Gish, which would give us the “phonetic” form in the Assyrian version dGish-gi-mash,60 as well as such a variant writing dGish-gi-mas-(si). Now a name like Gilgamesh, upon which we may definitely settle as coming closest to the genuine form, certainly impresses one as foreign, i.e., it is neither Sumerian nor Akkadian; and we have already suggested that the circumstance that the hero of the Epic is portrayed as a conqueror of Erech, and a rather ruthless one at that, points to a tradition of an invasion of the Euphrates Valley as the background for the episode in the first tablet of the series. Now it is significant that many of the names in the “mythical” dynasties, as they appear in Poebel’s list,61 are likewise foreign, such as Mes-ki-in-ga-še-ir, son of the god Shamash (and the founder of the “mythical” dynasty of Erech of which dGish-bil-ga-mesh is the fifth member),62 and En-me-ir-kár his son. In a still earlier “mythical” dynasty, we encounter names like Ga-lu-mu-um, Zu-ga-gi-ib, Ar-pi, [31]E-ta-na,63 which are distinctly foreign, while such names as En-me(n)-nun-na and Bar-sal-nun-na strike one again as “Sumerianized” names rather than as genuine Sumerian formations.64 Some of these names, as Galumum, Arpi and Etana, are so Amoritic in appearance, that one may hazard the conjecture of their western origin. May Gilgamesh likewise belong to the Amurru65 region, or does he represent a foreigner from the East in contrast to Enkidu, whose name, we have seen, may have been Baal-Ṭôb in the West, with which region he is according to the Epic so familiar? It must be confessed that the second element ga-mesh would fit in well with a Semitic origin for the name, for the element impresses one as the participial form of a Semitic stem g-m-š, just as in the second element of Meskin-gašer we have such a form. Gil might then be the name of a West-Semitic deity. Such conjectures, however, can for the present not be substantiated, and we must content ourselves with the conclusion that Gilgamesh as the real name of the hero, or at least the form which comes closest to the real name, points to a foreign origin for the hero, and that such forms as dGish-bil-ga-mesh and dGish-bíl-gi-mesh and other variants are “Sumerianized” forms for which an artificial etymology was brought forward to convey the [32]idea of the “original hero” or the hero par excellence. By means of this “play” on the name, which reverts to the compilers of the Sumerian version of the Epic, Gilgamesh was converted into a Sumerian figure, just as the name Enkidu may have been introduced as a Sumerian translation of his Amoritic name. dGish at all events is an abbreviated form of the “Sumerianized” name, introduced by the compilers of the earliest Akkadian version, which was produced naturally under the influence of the Sumerian version. Later, as the Epic continued to grow, a phonetic writing was introduced, dGish-gi-mash, which is in a measure a compromise between the genuine name and the “Sumerianized” form, but at the same time an approach to the real pronunciation. Next to the new light thrown upon the names and original character of the two main figures of the Epic, one of the chief points of interest in the Pennsylvania fragment is the proof that it furnishes for a striking resemblance of the two heroes, Gish and Enkidu, to one another. In interpreting the dream of Gish, his mother. Ninsun, lays stress upon the fact that the dream portends the coming of someone who is like Gish, “born in the field and reared in the mountain” (lines 18–19). Both, therefore, are shown by this description to have come to Babylonia from a mountainous region, i.e., they are foreigners; and in the case of Enkidu we have seen that the mountain in all probability refers to a region in the West, while the same may also be the case with Gish. The resemblance of the two heroes to one another extends to their personal appearance. When Enkidu appears on the streets of Erech, the people are struck by this resemblance. They remark that he is “like Gish,” though “shorter in stature” (lines 179–180). Enkidu is described as a rival or counterpart.66 This relationship between the two is suggested also by the Assyrian version. In the creation of Enkidu by Aruru, the people urge the goddess to create the “counterpart” (zikru) of Gilgamesh, someone who will be like him (ma-ši-il) (Tablet I, 2, 31). Enkidu not only comes from the mountain,67 but the mountain is specifically designated [33]as his birth-place (I, 4, 2), precisely as in the Pennsylvania tablet, while in another passage he is also described, as in our tablet, as “born in the field.”68 Still more significant is the designation of Gilgamesh as the talimu, “younger brother,” of Enkidu.69 In accord with this, we find Gilgamesh in his lament over Enkidu describing him as a “younger brother” (ku-ta-ni);70 and again in the last tablet of the Epic, Gilgamesh is referred to as the “brother” of Enkidu.71 This close relationship reverts to the Sumerian version, for the Constantinople fragment (Langdon, above, p. 13) begins with the designation of Gish-bil-ga-mesh as “his brother.” By “his” no doubt Enkidu is meant. Likewise in the Sumerian text published by Zimmern (above, p. 13) Gilgamesh appears as the brother of Enkidu (rev. 1, 17). Turning to the numerous representations of Gilgamesh and Enkidu on Seal Cylinders,72 we find this resemblance of the two heroes to each other strikingly confirmed. Both are represented as bearded, with the strands arranged in the same fashion. The face in both cases is broad, with curls protruding at the side of the head, though at times these curls are lacking in the case of Enkidu. What is particularly striking is to find Gilgamesh generally a little taller than Enkidu, thus bearing out the statement in the Pennsylvania tablet that Enkidu is “shorter in stature.” There are, to be sure, also some distinguishing marks between the two. Thus Enkidu is generally represented with animal hoofs, but not always.73 Enkidu is commonly portrayed with the horns of a bison, but again this sign is wanting in quite a number of instances.74 The hoofs and the horns mark the period when Enkidu lived with animals and much like an [34]animal. Most remarkable, however, of all are cylinders on which we find the two heroes almost exactly alike as, for example, Ward No. 199 where two figures, the one a duplicate of the other (except that one is just a shade taller), are in conflict with each other. Dr. Ward was puzzled by this representation and sets it down as a “fantastic” scene in which “each Gilgamesh is stabbing the other.” In the light of the Pennsylvania tablet, this scene is clearly the conflict between the two heroes described in column 6, preliminary to their forming a friendship. Even in the realm of myth the human experience holds good that there is nothing like a good fight as a basis for a subsequent alliance. The fragment describes this conflict as a furious one in which Gilgamesh is worsted, and his wounded pride assuaged by the generous victor, who comforts his vanquished enemy by the assurance that he was destined for something higher than to be a mere “Hercules.” He was singled out for the exercise of royal authority. True to the description of the two heroes in the Pennsylvania tablet as alike, one the counterpart of the other, the seal cylinder portrays them almost exactly alike, as alike as two brothers could possibly be; with just enough distinction to make it clear on close inspection that two figures are intended and not one repeated for the sake of symmetry. There are slight variations in the manner in which the hair is worn, and slightly varying expressions of the face, just enough to make it evident that the one is intended for Gilgamesh and the other for Enkidu. When, therefore, in another specimen, No. 173, we find a Gilgamesh holding his counterpart by the legs, it is merely another aspect of the fight between the two heroes, one of whom is intended to represent Enkidu, and not, as Dr. Ward supposed, a grotesque repetition of Gilgamesh.75 The description of Enkidu in the Pennsylvania tablet as a parallel figure to Gilgamesh leads us to a consideration of the relationship of the two figures to one another. Many years ago it was pointed out that the Gilgamesh Epic was a composite tale in which various stories of an independent origin had been combined and brought into more or less artificial connection with the heros eponymos of southern Babylonia.76 We may now go a step further and point out that not [35]only is Enkidu originally an entirely independent figure, having no connection with Gish or Gilgamesh, but that the latter is really depicted in the Epic as the counterpart of Enkidu, a reflection who has been given the traits of extraordinary physical power that belong to Enkidu. This is shown in the first place by the fact that in the encounter it is Enkidu who triumphs over Gilgamesh. The entire analysis of the episode of the meeting between the two heroes as given by Gressmann77 must be revised. It is not Enkidu who is terrified and who is warned against the encounter. It is Gilgamesh who, during the night on his way from the house in which the goddess Ishḫara lies, encounters Enkidu on the highway. Enkidu “blocks the path”78 of Gilgamesh. He prevents Gilgamesh from re-entering the house,79 and the two attack each other “like oxen.”80 They grapple with each other, and Enkidu forces Gilgamesh to the ground. Enkidu is, therefore, the real hero whose traits of physical prowess are afterwards transferred to Gilgamesh. Similarly in the next episode, the struggle against Ḫuwawa, the Yale tablet makes it clear that in the original form of the tale Enkidu is the real hero. All warn Gish against the undertaking—the elders of Erech, Enkidu, and also the workmen. “Why dost thou desire to do this?”81 they say to him. “Thou art young, and thy heart carries thee away. Thou knowest not what thou proposest to do.”82 This part of the incident is now better known to us through the latest fragment of the Assyrian version discovered and published by King.83 The elders say to Gilgamesh: “Do not trust, O Gilgamesh, in thy strength! Be warned(?) against trusting to thy attack! The one who goes before will save his companion,84 He who has foresight will save his friend.85 [36] Let Enkidu go before thee. He knows the roads to the cedar forest; He is skilled in battle and has seen fight.” Gilgamesh is sufficiently impressed by this warning to invite Enkidu to accompany him on a visit to his mother, Ninsun, for the purpose of receiving her counsel.86 It is only after Enkidu, who himself hesitates and tries to dissuade Gish, decides to accompany the latter that the elders of Erech are reconciled and encourage Gish for the fray. The two in concert proceed against Ḫuwawa. Gilgamesh alone cannot carry out the plan. Now when a tale thus associates two figures in one deed, one of the two has been added to the original tale. In the present case there can be little doubt that Enkidu, without whom Gish cannot proceed, who is specifically described as “acquainted with the way … to the entrance of the forest”87 in which Ḫuwawa dwells is the original vanquisher. Naturally, the Epic aims to conceal this fact as much as possible ad majorem gloriam of Gilgamesh. It tries to put the one who became the favorite hero into the foreground. Therefore, in both the Babylonian and the Assyrian version Enkidu is represented as hesitating, and Gilgamesh as determined to go ahead. Gilgamesh, in fact, accuses Enkidu of cowardice and boldly declares that he will proceed even though failure stare him in the face.88 Traces of the older view, however, in which Gilgamesh is the one for whom one fears the outcome, crop out; as, for example, in the complaint of Gilgamesh’s mother to Shamash that the latter has stirred the heart of her son to take the distant way to Ḫu(m)baba, “To a fight unknown to him, he advances, An expedition unknown to him he undertakes.”89 Ninsun evidently fears the consequences when her son informs her of his intention and asks her counsel. The answer of Shamash is not preserved, but no doubt it was of a reassuring character, as was the answer of the Sun-god to Gish’s appeal and prayer as set forth in the Yale tablet.90 [37] Again, as a further indication that Enkidu is the real conqueror of Ḫuwawa, we find the coming contest revealed to Enkidu no less than three times in dreams, which Gilgamesh interprets.91 Since the person who dreams is always the one to whom the dream applies, we may see in these dreams a further trace of the primary rôle originally assigned to Enkidu. Another exploit which, according to the Assyrian version, the two heroes perform in concert is the killing of a bull, sent by Anu at the instance of Ishtar to avenge an insult offered to the goddess by Gilgamesh, who rejects her offer of marriage. In the fragmentary description of the contest with the bull, we find Enkidu “seizing” the monster by “its tail.”92 That Enkidu originally played the part of the slayer is also shown by the statement that it is he who insults Ishtar by throwing a piece of the carcass into the goddess’ face,93 adding also an insulting speech; and this despite the fact that Ishtar in her rage accuses Gilgamesh of killing the bull.94 It is thus evident that the Epic alters the original character of the episodes in order to find a place for Gilgamesh, with the further desire to assign to the latter the chief rôle. Be it noted also that Enkidu, not Gilgamesh, is punished for the insult to Ishtar. Enkidu must therefore in the original form of the episode have been the guilty party, who is stricken with mortal disease as a punishment to which after twelve days he succumbs.95 In view of this, we may supply the name of Enkidu in the little song introduced at the close of the encounter with the bull, and not Gilgamesh as has hitherto been done. “Who is distinguished among the heroes? Who is glorious among men? [Enkidu] is distinguished among heroes, [Enkidu] is glorious among men.”96 [38]Finally, the killing of lions is directly ascribed to Enkidu in the Pennsylvania tablet: “Lions he attacked *     *     *     *     * Lions he overcame”97 whereas Gilgamesh appears to be afraid of lions. On his long search for Utnapishtim he says: “On reaching the entrance of the mountain at night I saw lions and was afraid.”98 He prays to Sin and Ishtar to protect and save him. When, therefore, in another passage some one celebrates Gilgamesh as the one who overcame the “guardian,” who dispatched Ḫu(m)baba in the cedar forest, who killed lions and overthrew the bull,99 we have the completion of the process which transferred to Gilgamesh exploits and powers which originally belonged to Enkidu, though ordinarily the process stops short at making Gilgamesh a sharer in the exploits; with the natural tendency, to be sure, to enlarge the share of the favorite. We can now understand why the two heroes are described in the Pennsylvania tablet as alike, as born in the same place, aye, as brothers. Gilgamesh in the Epic is merely a reflex of Enkidu. The latter is the real hero and presumably, therefore, the older figure.100 Gilgamesh resembles Enkidu, because he is originally Enkidu. The “resemblance” motif is merely the manner in which in the course of the partly popular, partly literary transfer, the recollection is preserved that Enkidu is the original, and Gilgamesh the copy. The artificiality of the process which brings the two heroes together is apparent in the dreams of Gilgamesh which are interpreted by his mother as portending the coming of Enkidu. Not the conflict is foreseen, but the subsequent close association, naïvely described as due to the personal charm which Enkidu exercises, which will lead Gilgamesh to fall in love with the one whom he is to meet. The two will become one, like man and wife. [39] On the basis of our investigations, we are now in a position to reconstruct in part the cycle of episodes that once formed part of an Enkidu Epic. The fight between Enkidu and Gilgamesh, in which the former is the victor, is typical of the kind of tales told of Enkidu. He is the real prototype of the Greek Hercules. He slays lions, he overcomes a powerful opponent dwelling in the forests of Lebanon, he kills the bull, and he finally succumbs to disease sent as a punishment by an angry goddess. The death of Enkidu naturally formed the close of the Enkidu Epic, which in its original form may, of course, have included other exploits besides those taken over into the Gilgamesh Epic. There is another aspect of the figure of Enkidu which is brought forward in the Pennsylvania tablet more clearly than had hitherto been the case. Many years ago attention was called to certain striking resemblances between Enkidu and the figure of the first man as described in the early chapters of Genesis.101 At that time we had merely the Assyrian version of the Gilgamesh Epic at our disposal, and the main point of contact was the description of Enkidu living with the animals, drinking and feeding like an animal, until a woman is brought to him with whom he engages in sexual intercourse. This suggested that Enkidu was a picture of primeval man, while the woman reminded one of Eve, who when she is brought to Adam becomes his helpmate and inseparable companion. The Biblical tale stands, of course, on a much higher level, and is introduced, as are other traditions and tales of primitive times, in the style of a parable to convey certain religious teachings. For all that, suggestions of earlier conceptions crop out in the picture of Adam surrounded by animals to which he assigns names. Such a phrase as “there was no helpmate corresponding to him” becomes intelligible on the supposition of an existing tradition or belief, that man once lived and, indeed, cohabited with animals. The tales in the early chapters of Genesis must rest on very early popular traditions, which have been cleared of mythological and other objectionable features in order to adapt them to the purpose of the Hebrew compilers, to serve as a medium for illustrating [40]certain religious teachings regarding man’s place in nature and his higher destiny. From the resemblance between Enkidu and Adam it does not, of course, follow that the latter is modelled upon the former, but only that both rest on similar traditions of the condition under which men lived in primeval days prior to the beginnings of human culture. We may now pass beyond these general indications and recognize in the story of Enkidu as revealed by the Pennsylvania tablet an attempt to trace the evolution of primitive man from low beginnings to the regular and orderly family life associated with advanced culture. The new tablet furnishes a further illustration for the surprisingly early tendency among the Babylonian literati to connect with popular tales teachings of a religious or ethical character. Just as the episode between Gilgamesh and the maiden Sabitum is made the occasion for introducing reflections on the inevitable fate of man to encounter death, so the meeting of Enkidu with the woman becomes the medium of impressing the lesson of human progress through the substitution of bread and wine for milk and water, through the institution of the family, and through work and the laying up of resources. This is the significance of the address to Enkidu in column 4 of the Pennsylvania tablet, even though certain expressions in it are somewhat obscure. The connection of the entire episode of Enkidu and the woman with Gilgamesh is very artificial; and it becomes much more intelligible if we disassociate it from its present entanglement in the Epic. In Gilgamesh’s dream, portending the meeting with Enkidu, nothing is said of the woman who is the companion of the latter. The passage in which Enkidu is created by Aruru to oppose Gilgamesh102 betrays evidence of having been worked over in order to bring Enkidu into association with the longing of the people of Erech to get rid of a tyrannical character. The people in their distress appeal to Aruru to create a rival to Gilgamesh. In response, “Aruru upon hearing this created a man of Anu in her heart.” Now this “man of Anu” cannot possibly be Enkidu, for the sufficient reason that a few lines further on Enkidu is described as an [41]offspring of Ninib. Moreover, the being created is not a “counterpart” of Gilgamesh, but an animal-man, as the description that follows shows. We must separate lines 30–33 in which the creation of the “Anu man” is described from lines 34–41 in which the creation of Enkidu is narrated. Indeed, these lines strike one as the proper beginning of the original Enkidu story, which would naturally start out with his birth and end with his death. The description is clearly an account of the creation of the first man, in which capacity Enkidu is brought forward. “Aruru washed her hands, broke off clay, threw it on the field103 … created Enkidu, the hero, a lofty offspring of the host of Ninib.”104 The description of Enkidu follows, with his body covered with hair like an animal, and eating and drinking with the animals. There follows an episode105 which has no connection whatsoever with the Gilgamesh Epic, but which is clearly intended to illustrate how Enkidu came to abandon the life with the animals. A hunter sees Enkidu and is amazed at the strange sight—an animal and yet a man. Enkidu, as though resenting his condition, becomes enraged at the sight of the hunter, and the latter goes to his father and tells him of the strange creature whom he is unable to catch. In reply, the father advises his son to take a woman with him when next he goes out on his pursuit, and to have the woman remove her dress in the presence of Enkidu, who will then approach her, and after intercourse with her will abandon the animals among whom he lives. By this device he will catch the strange creature. Lines 14–18 of column 3 in the first tablet in which the father of the hunter refers to Gilgamesh must be regarded as a later insertion, a part of the reconstruction of the tale to connect the episode with Gilgamesh. The advice of the father to his son, the hunter, begins, line 19, “Go my hunter, take with thee a woman.” [42]In the reconstructed tale, the father tells his son to go to Gilgamesh to relate to him the strange appearance of the animal-man; but there is clearly no purpose in this, as is shown by the fact that when the hunter does so, Gilgamesh makes precisely the same speech as does the father of the hunter. Lines 40–44 of column 3, in which Gilgamesh is represented as speaking to the hunter form a complete doublet to lines 19–24, beginning “Go, my hunter, take with thee a woman, etc.” and similarly the description of Enkidu appears twice, lines 2–12 in an address of the hunter to his father, and lines 29–39 in the address of the hunter to Gilgamesh. The artificiality of the process of introducing Gilgamesh into the episode is revealed by this awkward and entirely meaningless repetition. We may therefore reconstruct the first two scenes in the Enkidu Epic as follows:106 Tablet I, col. 2, 34–35: Creation of Enkidu by Aruru. 36–41: Description of Enkidu’s hairy body and of his life with the animals. 42–50: The hunter sees Enkidu, who shows his anger, as also his woe, at his condition. 3, 1–12: The hunter tells his father of the strange being who pulls up the traps which the hunter digs, and who tears the nets so that the hunter is unable to catch him or the animals. 19–24: The father of the hunter advises his son on his next expedition to take a woman with him in order to lure the strange being from his life with the animals. Line 25, beginning “On the advice of his father,” must have set forth, in the original form of the episode, how the hunter procured the woman and took her with him to meet Enkidu. Column 4 gives in detail the meeting between the two, and naïvely describes how the woman exposes her charms to Enkidu, who is captivated by her and stays with her six days and seven nights. The animals see the change in Enkidu and run away from him. [43]He has been transformed through the woman. So far the episode. In the Assyrian version there follows an address of the woman to Enkidu beginning (col. 4, 34): “Beautiful art thou, Enkidu, like a god art thou.” We find her urging him to go with her to Erech, there to meet Gilgamesh and to enjoy the pleasures of city life with plenty of beautiful maidens. Gilgamesh, she adds, will expect Enkidu, for the coming of the latter to Erech has been foretold in a dream. It is evident that here we have again the later transformation of the Enkidu Epic in order to bring the two heroes together. Will it be considered too bold if we assume that in the original form the address of the woman and the construction of the episode were such as we find preserved in part in columns 2 to 4 of the Pennsylvania tablet, which forms part of the new material that can now be added to the Epic? The address of the woman begins in line 51 of the Pennsylvania tablet: “I gaze upon thee, Enkidu, like a god art thou.” This corresponds to the line in the Assyrian version (I, 4, 34) as given above, just as lines 52–53: “Why with the cattle Dost thou roam across the field?” correspond to I, 4, 35, of the Assyrian version. There follows in both the old Babylonian and the Assyrian version the appeal of the woman to Enkidu, to allow her to lead him to Erech where Gilgamesh dwells (Pennsylvania tablet lines 54–61 = Assyrian version I, 4, 36–39); but in the Pennsylvania tablet we now have a second speech (lines 62–63) beginning like the first one with al-ka, “come:” “Come, arise from the accursed ground.” Enkidu consents, and now the woman takes off her garments and clothes the naked Enkidu, while putting another garment on herself. She takes hold of his hand and leads him to the sheepfolds (not to Erech!!), where bread and wine are placed before him. Accustomed hitherto to sucking milk with cattle, Enkidu does not know what to do with the strange food until encouraged and instructed by the woman. The entire third column is taken up with this introduction [44]of Enkidu to civilized life in a pastoral community, and the scene ends with Enkidu becoming a guardian of flocks. Now all this has nothing to do with Gilgamesh, and clearly sets forth an entirely different idea from the one embodied in the meeting of the two heroes. In the original Enkidu tale, the animal-man is looked upon as the type of a primitive savage, and the point of the tale is to illustrate in the naïve manner characteristic of folklore the evolution to the higher form of pastoral life. This aspect of the incident is, therefore, to be separated from the other phase which has as its chief motif the bringing of the two heroes together. We now obtain, thanks to the new section revealed by the Pennsylvania tablet, a further analogy107 with the story of Adam and Eve, but with this striking difference, that whereas in the Babylonian tale the woman is the medium leading man to the higher life, in the Biblical story the woman is the tempter who brings misfortune to man. This contrast is, however, not inherent in the Biblical story, but due to the point of view of the Biblical writer, who is somewhat pessimistically inclined and looks upon primitive life, when man went naked and lived in a garden, eating of fruits that grew of themselves, as the blessed life in contrast to advanced culture which leads to agriculture and necessitates hard work as the means of securing one’s substance. Hence the woman through whom Adam eats of the tree of knowledge and becomes conscious of being naked is looked upon as an evil tempter, entailing the loss of the primeval life of bliss in a gorgeous Paradise. The Babylonian point of view is optimistic. The change to civilized life—involving the wearing of clothes and the eating of food that is cultivated (bread and wine) is looked upon as an advance. Hence the woman is viewed as the medium of raising man to a higher level. The feature common to the Biblical and Babylonian tales is the attachment of a lesson to early folk-tales. The story of Adam and Eve,108 as the story of Enkidu and the woman, is told with a purpose. Starting with early traditions of men’s primitive life on earth, that may have arisen independently, Hebrew and [45]Babylonian writers diverged, each group going its own way, each reflecting the particular point of view from which the evolution of human society was viewed. Leaving the analogy between the Biblical and Babylonian tales aside, the main point of value for us in the Babylonian story of Enkidu and the woman is the proof furnished by the analysis, made possible through the Pennsylvania tablet, that the tale can be separated from its subsequent connection with Gilgamesh. We can continue this process of separation in the fourth column, where the woman instructs Enkidu in the further duty of living his life with the woman decreed for him, to raise a family, to engage in work, to build cities and to gather resources. All this is looked upon in the same optimistic spirit as marking progress, whereas the Biblical writer, consistent with his point of view, looks upon work as a curse, and makes Cain, the murderer, also the founder of cities. The step to the higher forms of life is not an advance according to the J document. It is interesting to note that even the phrase the “cursed ground” occurs in both the Babylonian and Biblical tales; but whereas in the latter (Gen. 3, 17) it is because of the hard work entailed in raising the products of the earth that the ground is cursed, in the former (lines 62–63) it is the place in which Enkidu lives before he advances to the dignity of human life that is “cursed,” and which he is asked to leave. Adam is expelled from Paradise as a punishment, whereas Enkidu is implored to leave it as a necessary step towards progress to a higher form of existence. The contrast between the Babylonian and the Biblical writer extends to the view taken of viniculture. The Biblical writer (again the J document) looks upon Noah’s drunkenness as a disgrace. Noah loses his sense of shame and uncovers himself (Genesis 9, 21), whereas in the Babylonian description Enkidu’s jolly spirit after he has drunk seven jars of wine meets with approval. The Biblical point of view is that he who drinks wine becomes drunk;109 the Babylonian says, if you drink wine you become happy.110 If the thesis here set forth of the original character and import of the episode of Enkidu with the woman is correct, we may again regard lines 149–153 of the Pennsylvania tablet, in which Gilgamesh is introduced, as a later addition to bring the two heroes into association. [46]The episode in its original form ended with the introduction of Enkidu first to pastoral life, and then to the still higher city life with regulated forms of social existence. Now, to be sure, this Enkidu has little in common with the Enkidu who is described as a powerful warrior, a Hercules, who kills lions, overcomes the giant Ḫuwawa, and dispatches a great bull, but it is the nature of folklore everywhere to attach to traditions about a favorite hero all kinds of tales with which originally he had nothing to do. Enkidu, as such a favorite, is viewed also as the type of primitive man,111 and so there arose gradually an Epic which began with his birth, pictured him as half-animal half-man, told how he emerged from this state, how he became civilized, was clothed, learned to eat food and drink wine, how he shaved off the hair with which his body was covered,112 anointed himself—in short, “He became manlike.”113 Thereupon he is taught his duties as a husband, is introduced to the work of building, and to laying aside supplies, and the like. The fully-developed and full-fledged hero then engages in various exploits, of which some are now embodied in the Gilgamesh Epic. Who this Enkidu was, we are not in a position to determine, but the suggestion has been thrown out above that he is a personage foreign to Babylonia, that his home appears to be in the undefined Amurru district, and that he conquers that district. The original tale of Enkidu, if this view be correct, must therefore have been carried to the Euphrates Valley, at a very remote period, with one of the migratory waves that brought a western people as invaders into Babylonia. Here the tale was combined with stories current of another hero, Gilgamesh—perhaps also of Western origin—whose conquest of Erech likewise represents an invasion of Babylonia. The center of the Gilgamesh tale was Erech, and in the process of combining the stories of Enkidu and Gilgamesh, Enkidu is brought to Erech and the two perform exploits [47]in common. In such a combination, the aim would be to utilize all the incidents of both tales. The woman who accompanies Enkidu, therefore, becomes the medium of bringing the two heroes together. The story of the evolution of primitive man to civilized life is transformed into the tale of Enkidu’s removal to Erech, and elaborated with all kinds of details, among which we have, as perhaps embodying a genuine historical tradition, the encounter of the two heroes. Before passing on, we have merely to note the very large part taken in both the old Babylonian and the Assyrian version by the struggle against Ḫuwawa. The entire Yale tablet—forming, as we have seen, the third of the series—is taken up with the preparation for the struggle, and with the repeated warnings given to Gilgamesh against the dangerous undertaking. The fourth tablet must have recounted the struggle itself, and it is not improbable that this episode extended into the fifth tablet, since in the Assyrian version this is the case. The elaboration of the story is in itself an argument in favor of assuming some historical background for it—the recollection of the conquest of Amurru by some powerful warrior; and we have seen that this conquest must be ascribed to Enkidu and not to Gilgamesh. If, now, Enkidu is not only the older figure but the one who is the real hero of the most notable episode in the Gilgamesh Epic; if, furthermore, Enkidu is the Hercules who kills lions and dispatches the bull sent by an enraged goddess, what becomes of Gilgamesh? What is left for him? In the first place, he is definitely the conqueror of Erech. He builds the wall of Erech,114 and we may assume that the designation of the city as Uruk supûri, “the walled Erech,”115 rests upon this tradition. He is also associated with the great temple Eanna, “the heavenly house,” in Erech. To Gilgamesh belongs also the unenviable tradition of having exercised his rule in Erech so harshly that the people are impelled to implore Aruru to create a rival who may rid [48]the district of the cruel tyrant, who is described as snatching sons and daughters from their families, and in other ways terrifying the population—an early example of “Schrecklichkeit.” Tablets II to V inclusive of the Assyrian version being taken up with the Ḫuwawa episode, modified with a view of bringing the two heroes together, we come at once to the sixth tablet, which tells the story of how the goddess Ishtar wooed Gilgamesh, and of the latter’s rejection of her advances. This tale is distinctly a nature myth. The attempt of Gressmann116 to find some historical background to the episode is a failure. The goddess Ishtar symbolizes the earth which woos the sun in the spring, but whose love is fatal, for after a few months the sun’s power begins to wane. Gilgamesh, who in incantation hymns is invoked in terms which show that he was conceived as a sun-god,117 recalls to the goddess how she changed her lovers into animals, like Circe of Greek mythology, and brought them to grief. Enraged at Gilgamesh’s insult to her vanity, she flies to her father Anu and cries for revenge. At this point the episode of the creation of the bull is introduced, but if the analysis above given is correct it is Enkidu who is the hero in dispatching the bull, and we must assume that the sickness with which Gilgamesh is smitten is the punishment sent by Anu to avenge the insult to his daughter. This sickness symbolizes the waning strength of the sun after midsummer is past. The sun recedes from the earth, and this was pictured in the myth as the sun-god’s rejection of Ishtar; Gilgamesh’s fear of death marks the approach of the winter season, when the sun appears to have lost its vigor completely and is near to death. The entire episode is, therefore, a nature myth, symbolical of the passing of spring to midsummer and then to the bare season. The myth has been attached to Gilgamesh as a favorite figure, and then woven into a pattern with the episode of Enkidu and the bull. The bull episode can be detached from the nature myth without any loss to the symbolism of the tale of Ishtar and Gilgamesh. As already suggested, with Enkidu’s death after this conquest of the bull the original Enkidu Epic came to an end. In order to connect Gilgamesh with Enkidu, the former is represented as sharing [49]in the struggle against the bull. Enkidu is punished with death, while Gilgamesh is smitten with disease. Since both shared equally in the guilt, the punishment should have been the same for both. The differentiation may be taken as an indication that Gilgamesh’s disease has nothing to do with the bull episode, but is merely part of the nature myth. Gilgamesh now begins a series of wanderings in search of the restoration of his vigor, and this motif is evidently a continuation of the nature myth to symbolize the sun’s wanderings during the dark winter in the hope of renewed vigor with the coming of the spring. Professor Haupt’s view is that the disease from which Gilgamesh is supposed to be suffering is of a venereal character, affecting the organs of reproduction. This would confirm the position here taken that the myth symbolizes the loss of the sun’s vigor. The sun’s rays are no longer strong enough to fertilize the earth. In accord with this, Gilgamesh’s search for healing leads him to the dark regions118 in which the scorpion-men dwell. The terrors of the region symbolize the gloom of the winter season. At last Gilgamesh reaches a region of light again, described as a landscape situated at the sea. The maiden in control of this region bolts the gate against Gilgamesh’s approach, but the latter forces his entrance. It is the picture of the sun-god bursting through the darkness, to emerge as the youthful reinvigorated sun-god of the spring. Now with the tendency to attach to popular tales and nature myths lessons illustrative of current beliefs and aspirations, Gilgamesh’s search for renewal of life is viewed as man’s longing for eternal life. The sun-god’s waning power after midsummer is past suggests man’s growing weakness after the meridian of life has been left behind. Winter is death, and man longs to escape it. Gilgamesh’s wanderings are used as illustration of this longing, and accordingly the search for life becomes also the quest for immortality. Can the precious boon of eternal life be achieved? Popular fancy created the figure of a favorite of the gods who had escaped a destructive deluge in which all mankind had perished.119 Gilgamesh hears [50]of this favorite and determines to seek him out and learn from him the secret of eternal life. The deluge story, again a pure nature myth, symbolical of the rainy season which destroys all life in nature, is thus attached to the Epic. Gilgamesh after many adventures finds himself in the presence of the survivor of the Deluge who, although human, enjoys immortal life among the gods. He asks the survivor how he came to escape the common fate of mankind, and in reply Utnapishtim tells the story of the catastrophe that brought about universal destruction. The moral of the tale is obvious. Only those singled out by the special favor of the gods can hope to be removed to the distant “source of the streams” and live forever. The rest of mankind must face death as the end of life. That the story of the Deluge is told in the eleventh tablet of the series, corresponding to the eleventh month, known as the month of “rain curse”120 and marking the height of the rainy season, may be intentional, just as it may not be accidental that Gilgamesh’s rejection of Ishtar is recounted in the sixth tablet, corresponding to the sixth month,121 which marks the end of the summer season. The two tales may have formed part of a cycle of myths, distributed among the months of the year. The Gilgamesh Epic, however, does not form such a cycle. Both myths have been artificially attached to the adventures of the hero. For the deluge story we now have the definite proof for its independent existence, through Dr. Poebel’s publication of a Sumerian text which embodies the tale,122 and without any reference [51]to Gilgamesh. Similarly, Scheil and Hilprecht have published fragments of deluge stories written in Akkadian and likewise without any connection with the Gilgamesh Epic.123 In the Epic the story leads to another episode attached to Gilgamesh, namely, the search for a magic plant growing in deep water, which has the power of restoring old age to youth. Utnapishtim, the survivor of the deluge, is moved through pity for Gilgamesh, worn out by his long wanderings. At the request of his wife, Utnapishtim decides to tell Gilgamesh of this plant, and he succeeds in finding it. He plucks it and decides to take it back to Erech so that all may enjoy the benefit, but on his way stops to bathe in a cool cistern. A serpent comes along and snatches the plant from him, and he is forced to return to Erech with his purpose unachieved. Man cannot hope, when old age comes on, to escape death as the end of everything. Lastly, the twelfth tablet of the Assyrian version of the Gilgamesh Epic is of a purely didactic character, bearing evidence of having been added as a further illustration of the current belief that there is no escape from the nether world to which all must go after life has come to an end. Proper burial and suitable care of the dead represent all that can be done in order to secure a fairly comfortable rest for those who have passed out of this world. Enkidu is once more introduced into this episode. His shade is invoked by Gilgamesh and rises up out of the lower world to give a discouraging reply to Gilgamesh’s request, “Tell me, my friend, tell me, my friend, The law of the earth which thou hast experienced, tell me,” The mournful message comes back: “I cannot tell thee, my friend, I cannot tell.” Death is a mystery and must always remain such. The historical Gilgamesh has clearly no connection with the figure introduced into [52]this twelfth tablet. Indeed, as already suggested, the Gilgamesh Epic must have ended with the return to Erech, as related at the close of the eleventh tablet. The twelfth tablet was added by some school-men of Babylonia (or perhaps of Assyria), purely for the purpose of conveying a summary of the teachings in regard to the fate of the dead. Whether these six episodes covering the sixth to the twelfth tablets, (1) the nature myth, (2) the killing of the divine bull, (3) the punishment of Gilgamesh and the death of Enkidu, (4) Gilgamesh’s wanderings, (5) the Deluge, (6) the search for immortality, were all included at the time that the old Babylonian version was compiled cannot, of course, be determined until we have that version in a more complete form. Since the two tablets thus far recovered show that as early as 2000 B.C. the Enkidu tale had already been amalgamated with the current stories about Gilgamesh, and the endeavor made to transfer the traits of the former to the latter, it is eminently likely that the story of Ishtar’s unhappy love adventure with Gilgamesh was included, as well as Gilgamesh’s punishment and the death of Enkidu. With the evidence furnished by Meissner’s fragment of a version of the old Babylonian revision and by our two tablets, of the early disposition to make popular tales the medium of illustrating current beliefs and the teachings of the temple schools, it may furthermore be concluded that the death of Enkidu and the punishment of Gilgamesh were utilized for didactic purposes in the old Babylonian version. On the other hand, the proof for the existence of the deluge story in the Hammurabi period and some centuries later, independent of any connection with the Gilgamesh Epic, raises the question whether in the old Babylonian version, of which our two tablets form a part, the deluge tale was already woven into the pattern of the Epic. At all events, till proof to the contrary is forthcoming, we may assume that the twelfth tablet of the Assyrian version, though also reverting to a Babylonian original, dates as the latest addition to the Epic from a period subsequent to 2000 B.C.; and that the same is probably the case with the eleventh tablet. To sum up, there are four main currents that flow together in the Gilgamesh Epic even in its old Babylonian form: (1) the adventures of a mighty warrior Enkidu, resting perhaps on a faint tradition [53]of the conquest of Amurru by the hero; (2) the more definite recollection of the exploits of a foreign invader of Babylonia by the name of Gilgamesh, whose home appears likewise to have been in the West;124 (3) nature myths and didactic tales transferred to Enkidu and Gilgamesh as popular figures; and (4) the process of weaving the traditions, exploits, myths and didactic tales together, in the course of which process Gilgamesh becomes the main hero, and Enkidu his companion. Furthermore, our investigation has shown that to Enkidu belongs the episode with the woman, used to illustrate the evolution of primitive man to the ways and conditions of civilized life, the conquest of Ḫuwawa in the land of Amurru, the killing of lions and also of the bull, while Gilgamesh is the hero who conquers Erech. Identified with the sun-god, the nature myth of the union of the sun with the earth and the subsequent separation of the two is also transferred to him. The wanderings of the hero, smitten with disease, are a continuation of the nature myth, symbolizing the waning vigor of the sun with the approach of the wintry season. The details of the process which led to making Gilgamesh the favorite figure, to whom the traits and exploits of Enkidu and of the sun-god are transferred, escape us, but of the fact that Enkidu is the older figure, of whom certain adventures were set forth in a tale that once had an independent existence, there can now be little doubt in the face of the evidence furnished by the two tablets of the old Babylonian version; just as the study of these tablets shows that in the combination of the tales of Enkidu and Gilgamesh, the former is the prototype of which Gilgamesh is the copy. If the two are regarded as brothers, as born in the same place, even resembling one another in appearance and carrying out their adventures in common, it is because in the process of combination Gilgamesh becomes the reflex of Enkidu. That Enkidu is not the figure created by Aruru to relieve Erech of its tyrannical ruler is also shown by the fact that Gilgamesh remains in control of Erech. It is to Erech that he returns when he fails of his purpose to learn the secret of escape from old age and death. Erech is, therefore, not relieved of the presence of the ruthless ruler through Enkidu. The “Man of Anu” formed by Aruru as a deliverer is confused in the course of the growth of the [54]Epic with Enkidu, the offspring of Ninib, and in this way we obtain the strange contradiction of Enkidu and Gilgamesh appearing first as bitter rivals and then as close and inseparable friends. It is of the nature of Epic compositions everywhere to eliminate unnecessary figures by concentrating on one favorite the traits belonging to another or to several others. The close association of Enkidu and Gilgamesh which becomes one of the striking features in the combination of the tales of these two heroes naturally recalls the “Heavenly Twins” motif, which has been so fully and so suggestively treated by Professor J. Rendell Harris in his Cult of the Heavenly Twins, (London, 1906). Professor Harris has conclusively shown how widespread the tendency is to associate two divine or semi-divine beings in myths and legends as inseparable companions125 or twins, like Castor and Pollux, Romulus and Remus,126 the Acvins in the Rig-Veda,127 Cain and Abel, Jacob and Esau in the Old Testament, the Kabiri of the Phoenicians,128 Herakles and Iphikles in Greek mythology, Ambrica and Fidelio in Teutonic mythology, Patollo and Potrimpo in old Prussian mythology, Cautes and Cautopates in Mithraism, Jesus and Thomas (according to the Syriac Acts of Thomas), and the various illustrations of “Dioscuri in Christian Legends,” set forth by Dr. Harris in his work under this title, which carries the motif far down into the period of legends about Christian Saints who appear in pairs, including the reference to such a pair in Shakespeare’s Henry V: “And Crispin Crispian shall ne’er go by From that day to the ending of the world.”—(Act, IV, 3, 57–58.) There are indeed certain parallels which suggest that Enkidu-Gilgamesh may represent a Babylonian counterpart to the “Heavenly [55]Twins.” In the Indo-Iranian, Greek and Roman mythology, the twins almost invariably act together. In unison they proceed on expeditions to punish enemies.129 But after all, the parallels are of too general a character to be of much moment; and moreover the parallels stop short at the critical point, for Gilgamesh though worsted is not killed by Enkidu, whereas one of the “Heavenly Twins” is always killed by the brother, as Abel is by Cain, and Iphikles by his twin brother Herakles. Even the trait which is frequent in the earliest forms of the “Heavenly Twins,” according to which one is immortal and the other is mortal, though applying in a measure to Enkidu who is killed by Ishtar, while Gilgamesh the offspring of a divine pair is only smitten with disease, is too unsubstantial to warrant more than a general comparison between the Enkidu-Gilgamesh pair and the various forms of the “twin” motif found throughout the ancient world. For all that, the point is of some interest that in the Gilgamesh Epic we should encounter two figures who are portrayed as possessing the same traits and accomplishing feats in common, which suggest a partial parallel to the various forms in which the twin-motif appears in the mythologies, folk-lore and legends of many nations; and it may be that in some of these instances the duplication is due, as in the case of Enkidu and Gilgamesh, to an actual transfer of the traits of one figure to another who usurped his place. In concluding this study of the two recently discovered tablets of the old Babylonian version of the Gilgamesh Epic which has brought us several steps further in the interpretation and in our understanding of the method of composition of the most notable literary production of ancient Babylonia, it will be proper to consider the literary relationship of the old Babylonian to the Assyrian version. We have already referred to the different form in which the names of the chief figures appear in the old Babylonian version, dGish as against dGish-gì(n)-mash, dEn-ki-dũ as against dEn-ki-dú, Ḫu-wa-wa as against Ḫu(m)-ba-ba. Erech appears as Uruk ribîtim, “Erech of [56]the Plazas,” as against Uruk supûri, “walled Erech” (or “Erech within the walls”), in the Assyrian version.130 These variations point to an independent recension for the Assyrian revision; and this conclusion is confirmed by a comparison of parallel passages in our two tablets with the Assyrian version, for such parallels rarely extend to verbal agreements in details, and, moreover, show that the Assyrian version has been elaborated. Beginning with the Pennsylvania tablet, column I is covered in the Assyrian version by tablet I, 5, 25, to 6, 33, though, as pointed out above, in the Assyrian version we have the anticipation of the dreams of Gilgamesh and their interpretation through their recital to Enkidu by his female companion, whereas in the old Babylonian version we have the dreams directly given in a conversation between Gilgamesh and his mother. In the anticipation, there would naturally be some omissions. So lines 4–5 and 12–13 of the Pennsylvania tablet do not appear in the Assyrian version, but in their place is a line (I, 5, 35), to be restored to ”[I saw him and like] a woman I fell in love with him.” which occurs in the old Babylonian version only in connection with the second dream. The point is of importance as showing that in the Babylonian version the first dream lays stress upon the omen of the falling meteor, as symbolizing the coming of Enkidu, whereas the second dream more specifically reveals Enkidu as a man,131 of whom Gilgamesh is instantly enamored. Strikingly variant lines, though conveying the same idea, are frequent. Thus line 14 of the Babylonian version reads “I bore it and carried it to thee” and appears in the Assyrian version (I, 5, 35b supplied from 6, 26) “I threw it (or him) at thy feet”132 [57]with an additional line in elaboration “Thou didst bring him into contact with me”133 which anticipates the speech of the mother (Line 41 = Assyrian version I, 6, 33). Line 10 of the Pennsylvania tablet has pa-ḫi-ir as against iz-za-az I, 5, 31. Line 8 has ik-ta-bi-it as against da-an in the Assyrian version I, 5, 29. More significant is the variant to line 9 “I became weak and its weight I could not bear” as against I, 5, 30. “Its strength was overpowering,134 and I could not endure its weight.” The important lines 31–36 are not found in the Assyrian version, with the exception of I, 6, 27, which corresponds to lines 33–34, but this lack of correspondence is probably due to the fact that the Assyrian version represents the anticipation of the dreams which, as already suggested, might well omit some details. As against this we have in the Assyrian version I, 6, 23–25, an elaboration of line 30 in the Pennsylvania tablet and taken over from the recital of the first dream. Through the Assyrian version I, 6, 31–32, we can restore the closing lines of column I of the Pennsylvania tablet, while with line 33 = line 45 of the Pennsylvania tablet, the parallel between the two versions comes to an end. Lines 34–43 of the Assyrian version (bringing tablet I to a close)135 represent an elaboration of the speech of Ninsun, followed by a further address of Gilgamesh to his mother, and by the determination of Gilgamesh to seek out Enkidu.136 Nothing of this sort appears to have been included in the old Babylonian version.[58]Our text proceeds with the scene between Enkidu and the woman, in which the latter by her charms and her appeal endeavors to lead Enkidu away from his life with the animals. From the abrupt manner in which the scene is introduced in line 43 of the Pennsylvania tablet, it is evident that this cannot be the first mention of the woman. The meeting must have been recounted in the first tablet, as is the case in the Assyrian version.137 The second tablet takes up the direct recital of the dreams of Gilgamesh and then continues the narrative. Whether in the old Babylonian version the scene between Enkidu and the woman was described with the same naïve details, as in the Assyrian version, of the sexual intercourse between the two for six days and seven nights cannot of course be determined, though presumably the Assyrian version, with the tendency of epics to become more elaborate as they pass from age to age, added some realistic touches. Assuming that lines 44–63 of the Pennsylvania tablet—the cohabitation of Enkidu and the address of the woman—is a repetition of what was already described in the first tablet, the comparison with the Assyrian version I, 4, 16–41, not only points to the elaboration of the later version, but likewise to an independent recension, even where parallel lines can be picked out. Only lines 46–48 of the Pennsylvania tablet form a complete parallel to line 21 of column 4 of the Assyrian version. The description in lines 22–32 of column 4 is missing, though it may, of course, have been included in part in the recital in the first tablet of the old Babylonian version. Lines 49–59 of the Pennsylvania tablet are covered by 33–39, the only slight difference being the specific mention in line 58 of the Pennsylvania tablet of Eanna, the temple in Erech, described as “the dwelling of Anu,” whereas in the Assyrian version Eanna is merely referred to as the “holy house” and described as “the dwelling of Anu and Ishtar,” where Ishtar is clearly a later addition. Leaving aside lines 60–61, which may be merely a variant (though independent) of line 39 of column 4 of the Assyrian version, we now have in the Pennsylvania tablet a second speech of the woman to Enkidu (not represented in the Assyrian version) beginning like the first one with alka, “Come” (lines 62–63), in which she asks Enkidu to leave the “accursed ground” in which he dwells. This speech, as the description which follows, extending into columns 3–4, [59]and telling how the woman clothed Enkidu, how she brought him to the sheep folds, how she taught him to eat bread and to drink wine, and how she instructed him in the ways of civilization, must have been included in the second tablet of the Assyrian version which has come down to us in a very imperfect form. Nor is the scene in which Enkidu and Gilgamesh have their encounter found in the preserved portions of the second (or possibly the third) tablet of the Assyrian version, but only a brief reference to it in the fourth tablet,138 in which in Epic style the story is repeated, leading up to the second exploit—the joint campaign of Enkidu and Gilgamesh against Ḫuwawa. This reference, covering only seven lines, corresponds to lines 192–231 of the Pennsylvania tablet; but the former being the repetition and the latter the original recital, the comparison to be instituted merely reveals again the independence of the Assyrian version, as shown in the use of kibsu, “tread” (IV, 2, 46), for šêpu, “foot” (l. 216), i-na-uš, “quake” (line 5C), as against ir-tu-tu (ll. 221 and 226). Such variants as dGish êribam ûl iddin (l. 217) against dGilgamesh ana šurûbi ûl namdin, (IV, 2, 47). and again iṣṣabtûma kima lîm “they grappled at the gate of the family house” (IV, 2, 48), against iṣṣabtûma ina bâb bît emuti, “they grappled at the gate of the family house” (IV, 2, 48), all point once more to the literary independence of the Assyrian version. The end of the conflict and the reconciliation of the two heroes is likewise missing in the Assyrian version. It may have been referred to at the beginning of column 3139 of Tablet IV. Coming to the Yale tablet, the few passages in which a comparison [60]may be instituted with the fourth tablet of the Assyrian version, to which in a general way it must correspond, are not sufficient to warrant any conclusions, beyond the confirmation of the literary independence of the Assyrian version. The section comprised within lines 72–89, where Enkidu’s grief at his friend’s decision to fight Ḫuwawa is described140, and he makes confession of his own physical exhaustion, may correspond to Tablet IV, column 4, of the Assyrian version. This would fit in with the beginning of the reverse, the first two lines of which (136–137) correspond to column 5 of the fourth tablet of the Assyrian version, with a variation “seven-fold fear”141 as against “fear of men” in the Assyrian version. If lines 138–139 (in column 4) of the Yale tablet correspond to line 7 of column 5 of Tablet IV of the Assyrian version, we would again have an illustration of the elaboration of the later version by the addition of lines 3–6. But beyond this we have merely the comparison of the description of Ḫuwawa “Whose roar is a flood, whose mouth is fire, and whose breath is death” which occurs twice in the Yale tablet (lines 110–111 and 196–197), with the same phrase in the Assyrian version Tablet IV, 5, 3—but here, as just pointed out, with an elaboration. Practically, therefore, the entire Yale tablet represents an addition to our knowledge of the Ḫuwawa episode, and until we are fortunate enough to discover more fragments of the fourth tablet of the Assyrian version, we must content ourselves with the conclusions reached from a comparison of the Pennsylvania tablet with the parallels in the Assyrian version. It may be noted as a general point of resemblance in the exterior form of the old Babylonian and Assyrian versions that both were inscribed on tablets containing six columns, three on the obverse and three on the reverse; and that the length of the tablets—an average of 40 to 50 lines—was about the same, thus revealing in the external form a conventiona1 size for the tablets in the older period, which was carried over into later times. [61] 1 See for further details of this royal library, Jastrow, Civilization of Babylonia and Assyria, p. 21 seq. 2 Das Babylonische Nimrodepos (Leipzig, 1884–1891), supplemented by Haupt’s article Die Zwölfte Tafel des Babylonischen Nimrodepos in BA I, pp. 48–79, containing the fragments of the twelfth tablet. The fragments of the Epic in Ashurbanapal’s library—some sixty—represent portions of several copies. Sin-liḳî-unnini—perhaps from Erech, since this name appears as that of a family in tablets from Erech (see Clay, Legal Documents from Erech, Index, p. 73)—is named in a list of texts (K 9717—Haupt’s edition No. 51, line 18) as the editor of the Epic, though probably he was not the only compiler. Since the publication of Haupt’s edition, a few fragments were added by him as an appendix to Alfred Jeremias Izdubar-Nimrod (Leipzig, 1891) Plates II–IV, and two more are embodied in Jensen’s transliteration of all the fragments in the Keilinschriftliche Bibliothek VI; pp. 116–265, with elaborate notes, pp. 421–531. Furthermore a fragment, obtained from supplementary excavations at Kouyunjik, has been published by L. W. King in his Supplement to the Catalogue of the Cuneiform Tablets in the Kouyunjik Collection of the British Cuneiform Tablets in the Kouyunjik Collection of the British Museum No. 56 and PSBA Vol. 36, pp. 64–68. Recently a fragment of the 6th tablet from the excavations at Assur has been published by Ebeling, Keilschrifttexte aus Assur Religiösen Inhalts No. 115, and one may expect further portions to turn up. The designation “Nimrod Epic” on the supposition that the hero of the Babylonian Epic is identical with Nimrod, the “mighty hunter” of Genesis 10, has now been generally abandoned, in the absence of any evidence that the Babylonian hero bore a name like [10n]Nimrod. For all that, the description of Nimrod as the “mighty hunter” and the occurrence of a “hunter” in the Babylonian Epic (Assyrian version Tablet I)—though he is not the hero—points to a confusion in the Hebrew form of the borrowed tradition between Gilgamesh and Nimrod. The latest French translation of the Epic is by Dhorme, Choix de Textes Religieux Assyro-Babyloniens (Paris, 1907), pp. 182–325; the latest German translation by Ungnad-Gressmann, Das Gilgamesch-Epos (Göttingen, 1911), with a valuable analysis and discussion. These two translations now supersede Jensen’s translation in the Keilinschriftliche Bibliothek, which, however, is still valuable because of the detailed notes, containing a wealth of lexicographical material. Ungnad also gave a partial translation in Gressmann-Ranke, Altorientalische Texte and Bilder I, pp. 39–61. In English, we have translations of substantial portions by Muss-Arnolt in Harper’s Assyrian and Babylonian Literature (New York, 1901), pp. 324–368; by Jastrow, Religion of Babylonia and Assyria (Boston, 1898), Chap. XXIII; by Clay in Light on the Old Testament from Babel, pp. 78–84; by Rogers in Cuneiform Parallels to the Old Testament, pp. 80–103; and most recently by Jastrow in Sacred Books and Early Literature of the East (ed. C. F. Horne, New York, 1917), Vol. I, pp. 187–220. 3 See Luckenbill in JAOS, Vol. 37, p. 452 seq. Prof. Clay, it should be added, clings to the older reading, Hammurabi, which is retained in this volume. 4 ZA, Vol. 14, pp. 277–292. 5 The survivor of the Deluge is usually designated as Ut-napishtim in the Epic, but in one passage (Assyrian version, Tablet XI, 196), he is designated as Atra-ḫasis “the very wise one.” Similarly, in a second version of the Deluge story, also found in Ashurbanapal’s library (IV R² additions, p. 9, line 11). The two names clearly point to two versions, which in accordance with the manner of ancient compositions were merged into one. See an article by Jastrow in ZA, Vol. 13, pp. 288–301. 6 Published by Scheil in Recueil des Travaux, etc. Vol. 20, pp. 55–58. 7 The text does not form part of the Gilgamesh Epic, as the colophon, differing from the one attached to the Epic, shows. 8 Ein altbabylonisches Fragment des Gilgamosepos (MVAG 1902, No. 1). 9 On these variant forms of the two names see the discussion below, p. 24. 10 The passage is paralleled by Ecc. 9, 7–9. See Jastrow, A Gentle Cynic, p. 172 seq. 11 Among the Nippur tablets in the collection of the University of Pennsylvania Museum. The fragment was published by Dr. Poebel in his Historical and Grammatical Texts No. 23. See also Poebel in the Museum Journal, Vol. IV, p. 47, and an article by Dr. Langdon in the same Journal, Vol. VII, pp. 178–181, though Langdon fails to credit Dr. Poebel with the discovery and publication of the important tablet. 12 No. 55 in Langdon’s Historical and Religious Texts from the Temple Library of Nippur (Munich, 1914). 13 No. 5 in his Sumerian Liturgical Texts. (Philadelphia, 1917) 14 See on this name below, p. 23. 15 See further below, p. 37 seq. 16 See Poebel, Historical and Grammatical Texts, No. 1, and Jastrow in JAOS, Vol. 36, pp. 122–131 and 274–299. 17 See an article by Jastrow, Sumerian and Akkadian Views of Beginnings (JAOS Vol. 36, pp. 274–299). 18 See on this point Eduard Meyer, Sumerier und Semiten in Babylonien (Berlin, 1906), p. 107 seq., whose view is followed in Jastrow, Civilization of Babylonia and Assyria, p. 121. See also Clay, Empire of the Amorites (Yale University Press, 1919), p. 23 et seq. 19 See the discussion below, p. 24 seq. 20 Dr. Poebel published an article on the tablet in OLZ, 1914, pp. 4–6, in which he called attention to the correct name for the mother of Gilgamesh, which was settled by the tablet as Ninsun. 21 Historical Texts No. 2, Column 2, 26. See the discussion in Historical and Grammatical Texts, p. 123, seq. 22 See Fostat in OLZ, 1915, p. 367. 23 Publications of the University of Pennsylvania Museum, Babylonian Section, Vol. X, No. 3 (Philadelphia, 1917). It is to be regretted that Dr. Langdon should not have given full credit to Dr. Poebel for his discovery of the tablet. He merely refers in an obscure footnote to Dr. Poebel’s having made a copy. 24 E.g., in the very first note on page 211, and again in a note on page 213. 25 Dr. Langdon neglected to copy the signs 4 šú-si = 240 which appear on the edge of the tablet. He also misunderstood the word šú-tu-ur in the colophon which he translated “written,” taking the word from a stem šaṭâru, “write.” The form šú-tu-ur is III, 1, from atâru, “to be in excess of,” and indicates, presumably, that the text is a copy “enlarged” from an older original. See the Commentary to the colophon, p. 86. 26 Museum Journal, Vol. VIII, p. 29. 27 See below, p. 23. 28 I follow the enumeration of tablets, columns and lines in Jensen’s edition, though some fragments appear to have been placed by him in a wrong position. 29 According to Bezold’s investigation, Verbalsuffixformen als Alterskriterien babylonisch-assyrischer Inschriften (Heidelberg Akad. d. Wiss., Philos.-Histor. Klasse, 1910, 9te Abhandlung), the bulk of the tablets in Ashurbanapal’s library are copies of originals dating from about 1500 B.C. It does not follow, however, that all the copies date from originals of the same period. Bezold reaches the conclusion on the basis of various forms for verbal suffixes, that the fragments from the Ashurbanapal Library actually date from three distinct periods ranging from before c. 1450 to c. 700 B.C. 30 “Before thou comest from the mountain, Gilgamesh in Erech will see thy dreams,” after which the dreams are recounted by the woman to Enkidu. The expression “thy dreams” means here “dreams about thee.” (Tablet I, 5, 23–24). 31 Lines 100–101. 32 In a paper read before the American Oriental Society at New Haven, April 4, 1918. 33 See the commentary to col. 4 of the Yale tablet for further details. 34 This is no doubt the correct reading of the three signs which used to be read Iz-tu-bar or Gish-du-bar. The first sign has commonly the value Gish, the second can be read Gin or Gi (Brünnow No. 11900) and the third Mash as well as Bar. See Ungnad in Ungnad-Gressmann, Das Gilgamesch-Epos, p. 76, and Poebel, Historical and Grammatical Texts, p. 123. 35 So also in Sumerian (Zimmern, Sumerische Kultlieder aus altbabylonischer Zeit, No. 196, rev. 14 and 16.) 36 The sign used, LUM (Brünnow No. 11183), could have the value ḫu as well as ḫum. 37 The addition “father-in-law of Moses” to the name Ḫobab b. Re’uel in this passage must refer to Re’uel, and not to Ḫobab. In Judges 4, 11, the gloss “of the Bene Ḫobab, the father-in-law of Moses” must be separated into two: (1) “Bene Ḫobab,” and (2) “father-in-law of Moses.” The latter addition rests on an erroneous tradition, or is intended as a brief reminder that Ḫobab is identical with the son of Re’uel. 38 See his List of Personal Names from the Temple School of Nippur, p. 122. Ḫu-um-ba-bi-tu and ši-kin ḫu-wa-wa also occur in Omen Texts (CT XXVII, 4, 8–9 = Pl. 3, 17 = Pl. 6, 3–4 = CT XXVIII, 14, 12). The contrast to ḫuwawa is ligru, “dwarf” (CT XXVII, 4, 12 and 14 = Pl. 6, 7.9 = Pl. 3, 19). See Jastrow, Religion Babyloniens und Assyriens, II, p. 913, Note 7. Ḫuwawa, therefore, has the force of “monster.” 39 Ungnad-Gressmann, Das Gilgamesch-Epos, p. 111 seq. 40 Ungnad, 1. c. p. 77, called attention to this name, but failed to draw the conclusion that Ḫu(m)baba therefore belongs to the West and not to the East. 41 First pointed out by Ungnad in OLZ 1910, p. 306, on the basis of CT XVIII, 30, 10, where En-gi-dú appears in the column furnishing phonetic readings. 42 See Clay Amurru, pp. 74, 129, etc. 43 Tablet I, 2, 39–40; 3, 6–7 and 33–34; 4, 3–4. 44 Tablet I, 2, 1 and IX, 2, 16. Note also the statement about Gilgamesh that “his body is flesh of the gods” (Tablet IX, 2, 14; X, 1, 7). 45 BOR IV, p. 264. 46 Lewin, Die Scholien des Theodor bar Koni zur Patriarchengeschichte (Berlin, 1905), p. 2. See Gressmann in Ungnad-Gressmann, Das Gilgamesch-Epos, p. 83, who points out that the first element of גלמגוס compared with the second of גמיגמוס gives the exact form that we require, namely, Gilgamos. 47 Tablet I, col. 2, is taken up with this episode. 48 See Poebel, Historical and Grammatical Texts, p. 123. 49 See Poebel, Historical Texts No. 2, col. 2, 26. 50 Hilprecht, Old Babylonian Inscriptions I, 1 No. 26. 51 Delitzsch, Assyrische Lesestücke, p. 88, VI, 2–3. Cf. also CT XXV, 28(K 7659) 3, where we must evidently supply [Esigga]-tuk, for which in the following line we have again Gish-bil-ga-mesh as an equivalent. See Meissner, OLZ 1910, 99. 52 See, e.g., Barton, Haverford Collection II No. 27, Col. I, 14, etc. 53 Deimel, Pantheon Babylonicum, p. 95. 54 CT XII, 50 (K 4359) obv. 17. 55 See Barton, Origin and Development of Babylonian Writing, II, p. 99 seq., for various explanations, though all centering around the same idea of the picture of fire in some form. 56 See the passages quoted by Poebel, Historical and Grammatical Texts, p. 126. 57 E.g., Genesis 4, 20, Jabal, “the father of tent-dwelling and cattle holding;” Jubal (4, 21), “the father of harp and pipe striking.” 58 See particularly the plays (in the J. Document) upon the names of the twelve sons of Jacob, which are brought forward either as tribal characteristics, or as suggested by some incident or utterance by the mother at the birth of each son. 59 The designation is variously explained by Arabic writers. See Beidhawi’s Commentary (ed. Fleischer), to Súra 18, 82. 60 The writing Gish-gi-mash as an approach to the pronunciation Gilgamesh would thus represent the beginning of the artificial process which seeks to interpret the first syllable as “hero.” 61 See above, p. 27. 62 Poebel, Historical Texts, p. 115 seq. 63 Many years ago (BA III, p. 376) I equated Etana with Ethan in the Old Testament—therefore a West Semitic name. 64 See Clay, The Empire of the Amorites, p. 80. 65 Professor Clay strongly favors an Amoritic origin also for Gilgamesh. His explanation of the name is set forth in his recent work on The Empire of the Amorites, page 89, and is also referred to in his work on Amurru, page 79, and in his volume of Miscellaneous Inscriptions in the Yale Babylonian Collection, page 3, note. According to Professor Clay the original form of the hero’s name was West Semitic, and was something like Bilga-Mash, the meaning of which was perhaps “the offspring of Mash.” For the first element in this division of the name cf. Piliḳam, the name of a ruler of an early dynasty, and Balaḳ of the Old Testament. In view of the fact that the axe figures so prominently in the Epic as an instrument wielded by Gilgamesh, Professor Clay furthermore thinks it reasonable to assume that the name was interpreted by the Babylonian scribe as “the axe of Mash.” In this way he would account for the use of the determinative for weapons, which is also the sign Gish, in the name. It is certainly noteworthy that the ideogram Gish-Tún in the later form of Gish-Tún-mash = pašu, “axe,” CT XVI, 38:14b, etc. Tun also = pilaḳu “axe,” CT xii, 10:34b. Names with similar element (besides Piliḳam) are Belaḳu of the Hammurabi period, Bilaḳḳu of the Cassite period, etc. It is only proper to add that Professor Jastrow assumes the responsibility for the explanation of the form and etymology of the name Gilgamesh proposed in this volume. The question is one in regard to which legitimate differences of opinion will prevail among scholars until through some chance a definite decision, one way or the other, can be reached. 66 me-iḫ-rù (line 191). 67 Tablet I, 5, 23. Cf. I, 3, 2 and 29. 68 Tablet IV, 4, 7 and I, 5, 3. 69 Assyrian version, Tablet II, 3b 34, in an address of Shamash to Enkidu. 70 So Assyrian version, Tablet VIII, 3, 11. Also supplied VIII, 5, 20 and 21; and X, 1, 46–47 and 5, 6–7. 71 Tablet XII, 3, 25. 72 Ward, Seal Cylinders of Western Asia, Chap. X, and the same author’s Cylinders and other Ancient Oriental Seals—Morgan collection Nos. 19–50. 73 E.g., Ward No. 192, Enkidu has human legs like Gilgamesh; also No. 189, where it is difficult to say which is Gilgamesh, and which is Enkidu. The clothed one is probably Gilgamesh, though not infrequently Gilgamesh is also represented as nude, or merely with a girdle around his waist. 74 E.g., Ward, Nos. 173, 174, 190, 191, 195 as well as 189 and 192. 75 On the other hand, in Ward Nos. 459 and 461, the conflict between the two heroes is depicted with the heroes distinguished in more conventional fashion, Enkidu having the hoofs of an animal, and also with a varying arrangement of beard and hair. 76 See Jastrow, Religion of Babylonia and Assyria (Boston, 1898), p. 468 seq. 77 Ungnad-Gressmann, Das Gilgamesch-Epos, p. 90 seq. 78 Pennsylvania tablet, l. 198 = Assyrian version, Tablet IV, 2, 37. 79 “Enkidu blocked the gate” (Pennsylvania tablet, line 215) = Assyrian version Tablet IV, 2, 46: “Enkidu interposed his foot at the gate of the family house.” 80 Pennsylvania tablet, lines 218 and 224. 81 Yale tablet, line 198; also to be supplied lines 13–14. 82 Yale tablet, lines 190 and 191. 83 PSBA 1914, 65 seq. = Jensen III, 1a, 4–11, which can now be completed and supplemented by the new fragment. 84 I.e., Enkidu will save Gilgamesh. 85 These two lines impress one as popular sayings—here applied to Enkidu. 86 King’s fragment, col. I, 13–27, which now enables us to complete Jensen III, 1a, 12–21. 87 Yale tablet, lines 252–253. 88 Yale tablet, lines 143–148 = Assyrian version, Tablet IV, 6, 26 seq. 89 Assyrian version, Tablet III, 2a, 13–14. 90 Lines 215–222. 91 Assyrian version, Tablet V, Columns 3–4. We have to assume that in line 13 of column 4 (Jensen, p. 164), Enkidu takes up the thread of conversation, as is shown by line 22: “Enkidu brought his dream to him and spoke to Gilgamesh.” 92 Assyrian version, Tablet VI, lines 146–147. 93 Lines 178–183. 94 Lines 176–177. 95 Tablet VII, Column 6. 96 Assyrian version, Tablet VI, 200–203. These words are put into the mouth of Gilgamesh (lines 198–199). It is, therefore, unlikely that he would sing his own praise. Both Jensen and Ungnad admit that Enkidu is to be supplied in at least one of the lines. 97 Lines 109 and 112. 98 Assyrian version, Tablet IX, 1, 8–9. 99 Tablet VIII, 5, 2–6. 100 So also Gressmann in Ungnad-Gressmann, Das Gilgamesch-Epos, p. 97, regards Enkidu as the older figure. 101 See Jastrow, Adam and Eve in Babylonian Literature, AJSL, Vol. 15, pp. 193–214. 102 Assyrian version, Tablet I, 2, 31–36. 103 It will be recalled that Enkidu is always spoken of as “born in the field.” 104 Note the repetition ibtani “created” in line 33 of the “man of Anu” and in line 35 of the offspring of Ninib. The creation of the former is by the “heart,” i.e., by the will of Aruru, the creation of the latter is an act of moulding out of clay. 105 Tablet I, Column 3. 106 Following as usual the enumeration of lines in Jensen’s edition. 107 An analogy does not involve a dependence of one tale upon the other, but merely that both rest on similar traditions, which may have arisen independently. 108 Note that the name of Eve is not mentioned till after the fall (Genesis 3, 20). Before that she is merely ishsha, i.e., “woman,” just as in the Babylonian tale the woman who guides Enkidu is ḫarimtu, “woman.” 109 “And he drank and became drunk” (Genesis 9, 21). 110 “His heart became glad and his face shone” (Pennsylvania Tablet, lines 100–101). 111 That in the combination of this Enkidu with tales of primitive man, inconsistent features should have been introduced, such as the union of Enkidu with the woman as the beginning of a higher life, whereas the presence of a hunter and his father shows that human society was already in existence, is characteristic of folk-tales, which are indifferent to details that may be contradictory to the general setting of the story. 112 Pennsylvania tablet, lines 102–104. 113 Line 105. 114 Tablet I, 1, 9. See also the reference to the wall of Erech as an “old construction” of Gilgamesh, in the inscription of An-Am in the days of Sin-gamil (Hilprecht, Old Babylonian Inscriptions, I, No. 26.) Cf IV R² 52, 3, 53. 115 The invariable designation in the Assyrian version as against Uruk ribîtim, “Erech of the plazas,” in the old Babylonian version. 116 In Ungnad-Gressmann, Das Gilgamesch-Epos, p. 123 seq. 117 See Jensen, p. 266. Gilgamesh is addressed as “judge,” as the one who inspects the divisions of the earth, precisely as Shamash is celebrated. In line 8 of the hymn in question, Gilgamesh is in fact addressed as Shamash. 118 The darkness is emphasized with each advance in the hero’s wanderings (Tablet IX, col. 5). 119 This tale is again a nature myth, marking the change from the dry to the rainy season. The Deluge is an annual occurrence in the Euphrates Valley through the overflow [50n]of the two rivers. Only the canal system, directing the overflow into the fields, changed the curse into a blessing. In contrast to the Deluge, we have in the Assyrian creation story the drying up of the primeval waters so that the earth makes its appearance with the change from the rainy to the dry season. The world is created in the spring, according to the Akkadian view which is reflected in the Biblical creation story, as related in the P. document. See Jastrow, Sumerian and Akkadian Views of Beginnings (JAOS, Vol 36, p. 295 seq.). 120 Aš-am in Sumerian corresponding to the Akkadian Šabaṭu, which conveys the idea of destruction. 121 The month is known as the “Mission of Ishtar” in Sumerian, in allusion to another nature myth which describes Ishtar’s disappearance from earth and her mission to the lower world. 122 Historical Texts No. 1. The Sumerian name of the survivor is Zi-ū-gíd-du or perhaps Zi-ū-sū-du (cf. King, Legends of Babylon and Egypt, p. 65, note 4), signifying “He who lengthened the day of life,” i.e., the one of long life, of which Ut-napishtim (“Day of Life”) in the Assyrian version seems to be an abbreviated Akkadian rendering, [n]with the omission of the verb. So King’s view, which is here followed. See also CT XVIII, 30, 9, and Langdon, Sumerian Epic of Paradise, p. 90, who, however, enters upon further speculations that are fanciful. 123 See the translation in Ungnad-Gressmann, Das Gilgamesch-Epos, pp. 69, seq. and 73. 124 According to Professor Clay, quite certainly Amurru, just as in the case of Enkidu. 125 Gressmann in Ungnad-Gressmann, Das Gilgamesch-Epos, p. 100 seq. touches upon this motif, but fails to see the main point that the companions are also twins or at least brothers. Hence such examples as Abraham and Lot, David and Jonathan, Achilles and Patroclus, Eteokles and Polyneikes, are not parallels to Gilgamesh-Enkidu, but belong to the enlargement of the motif so as to include companions who are not regarded as brothers. 126 Or Romus. See Rendell Harris, l. c., p. 59, note 2. 127 One might also include the primeval pair Yama-Yami with their equivalents in Iranian mythology (Carnoy, Iranian Mythology, p. 294 seq.). 128 Becoming, however, a triad and later increased to seven. Cf. Rendell Harris, l. c., p. 32. 129 I am indebted to my friend, Professor A. J. Carnoy, of the University of Louvain, for having kindly gathered and placed at my disposal material on the “twin-brother” motif from Indo-European sources, supplemental to Rendell Harris’ work. 130 On the other hand, Uruk mâtum for the district of Erech, i.e., the territory over which the city holds sway, appears in both versions (Pennsylvania tablet, 1. 10 = Assyrian version I, 5, 36). 131 “My likeness” (line 27). It should be noted, however, that lines 32–44 of I, 5, in Jensen’s edition are part of a fragment K 9245 (not published, but merely copied by Bezold and Johns, and placed at Jensen’s disposal), which may represent a duplicate to I, 6, 23–34, with which it agrees entirely except for one line, viz., line 34 of K 9245 which is not found in column 6, 23–34. If this be correct, then there is lacking after line 31 of column 5, the interpretation of the dream given in the Pennsylvania tablet in lines 17–23. 132 ina šap-li-ki, literally, “below thee,” whereas in the old Babylonian version we have ana ṣi-ri-ka, “towards thee.” 133 Repeated I, 6, 28. 134 ul-tap-rid ki-is-su-šú-ma. The verb is from parâdu, “violent.” For kissu, “strong,” see CT XVI, 25, 48–49. Langdon (Gilgamesh Epic, p. 211, note 5) renders the phrase: “he shook his murderous weapon!!”—another illustration of his haphazard way of translating texts. 135 Shown by the colophon (Jeremias, Izdubar-Nimrod, Plate IV.) 136 Lines 42–43 must be taken as part of the narrative of the compiler, who tells us that after the woman had informed Enkidu that Gilgamesh already knew of Enkidu’s coming through dreams interpreted by Ninsun, Gilgamesh actually set out and encountered Enkidu. 137 Tablet I, col. 4. See also above, p. 19. 138 IV, 2, 44–50. The word ullanum, (l.43) “once” or “since,” points to the following being a reference to a former recital, and not an original recital. 139 Only the lower half (Haupt’s edition, p. 82) is preserved. 140 “The eyes of Enkidu were filled with tears,” corresponding to IV, 4, 10. 141 Unless indeed the number “seven” is a slip for the sign ša. See the commentary to the line. Pennsylvania Tablet The 240 lines of the six columns of the text are enumerated in succession, with an indication on the margin where a new column begins. This method, followed also in the case of the Yale tablet, seems preferable to Langdon’s breaking up of the text into Obverse and Reverse, with a separate enumeration for each of the six columns. In order, however, to facilitate a comparison with Langdon’s edition, a table is added: Obverse Col. I, 1 = Line 1 of our text. ,, I, 5 = ,, 5 ,, ,, ,, ,, I, 10 = ,, 10 ,, ,, ,, ,, I, 15 = ,, 15 ,, ,, ,, ,, I, 20 = ,, 20 ,, ,, ,, ,, I, 25 = ,, 25 ,, ,, ,, ,, I, 30 = ,, 30 ,, ,, ,, ,, I, 35 = ,, 35 ,, ,, ,, Col. II, 1 = Line 41 ,, ,, ,, ,, II, 5 = ,, 45 ,, ,, ,, ,, II, 10 = ,, 50 ,, ,, ,, ,, II, 15 = ,, 55 ,, ,, ,, ,, II, 20 = ,, 60 ,, ,, ,, ,, II, 25 = ,, 65 ,, ,, ,, ,, II, 30 = ,, 70 ,, ,, ,, ,, II, 35 = ,, 75 ,, ,, ,, Col. III, 1 = Line 81 ,, ,, ,, ,, III, 5 = ,, 85 ,, ,, ,, ,, III, 10 = ,, 90 ,, ,, ,, ,, III, 15 = ,, 95 ,, ,, ,, ,, III, 26 = ,, 100 ,, ,, ,, ,, III, 25 = ,, 105 ,, ,, ,, ,, III, 30 = ,, 110 ,, ,, ,, ,, III, 35 = ,, 115 ,, ,, ,, Reverse Col. I, 1 (= Col. IV) = Line 131 of our text. ,, I, 5 = ,, 135 ,, ,, ,, ,, I, 10 = ,, 140 ,, ,, ,, ,, I, 15 = ,, 145 ,, ,, ,, ,, I, 20 = ,, 150 ,, ,, ,, ,, I, 25 = ,, 155 ,, ,, ,, ,, I, 30 = ,, 160 ,, ,, ,, ,, II, 1 (= Col. V) = Line 171 ,, ,, ,, ,, II, 5 = ,, 175 ,, ,, ,, ,, II, 10 = ,, 180 ,, ,, ,, ,, II, 15 = ,, 185 ,, ,, ,, ,, II, 20 = ,, 190 ,, ,, ,, ,, II, 25 = ,, 195 ,, ,, ,, ,, II, 30 = ,, 200 ,, ,, ,, ,, III, 1 (= Col. VI) = Line 208 ,, ,, ,, ,, III, 5 = ,, 212 ,, ,, ,, ,, III, 10 = ,, 217 ,, ,, ,, ,, III, 15 = ,, 222 ,, ,, ,, ,, III, 20 = ,, 227 ,, ,, ,, ,, III, 25 = ,, 232 ,, ,, ,, ,, III, 30 = ,, 237 ,, ,, ,, ,, III, 33 = ,, 240 ,, ,, ,, [62] Pennsylvania Tablet. Transliteration. Col. I. 1it-bi-e-ma dGiš šú-na-tam i-pa-áš-šar 2iz-za-kàr-am a-na um-mi-šú 3um-mi i-na šá-at mu-ši-ti-ia 4šá-am-ḫa-ku-ma at-ta-na-al-la-ak 5i-na bi-ri-it it-lu-tim 6ib-ba-šú-nim-ma ka-ka-bu šá-ma-i 7[ki]-iṣ-rù šá A-nim im-ḳu-ut a-na ṣi-ri-ia 8áš-ši-šú-ma ik-ta-bi-it e-li-ia 9ú-ni-iš-šú-ma nu-uš-šá-šú ú-ul il-ti-’i 10Urukki ma-tum pa-ḫi-ir e-li-šú 11it-lu-tum ú-na-šá-ku ši-pi-šú 12ú-um-mi-id-ma pu-ti 13i-mi-du ia-ti 14áš-ši-a-šú-ma ab-ba-la-áš-šú a-na ṣi-ri-ki 15um-mi dGiš mu-di-a-at ka-la-ma 16iz-za-kàr-am a-na dGiš 17mi-in-di dGiš šá ki-ma ka-ti 18i-na ṣi-ri i-wa-li-id-ma 19ú-ra-ab-bi-šú šá-du-ú 20ta-mar-šú-ma [kima Sal(?)] ta-ḫa-du at-ta 21it-lu-tum ú-na-šá-ku ši-pi-šú 22tí-iṭ-ṭi-ra-áš-[šú tu-ut]-tu-ú-ma 23ta-tar-ra-[as-su] a-na ṣi-[ri]-ia 24[uš]-ti-nim-ma i-ta-mar šá-ni-tam[63] 25[šú-na]-ta i-ta-wa-a-am a-na um-mi-šú 26[um-mi] a-ta-mar šá-ni-tam 27[šú-na-tu a-ta]-mar e-mi-a i-na su-ḳi-im 28[šá Uruk]ki ri-bi-tim 29ḫa-aṣ-ṣi-nu na-di-i-ma 30e-li-šú pa-aḫ-ru 31ḫa-aṣ-ṣi-nu-um-ma šá-ni bu-nu-šú 32a-mur-šú-ma aḫ-ta-du a-na-ku 33a-ra-am-šú-ma ki-ma áš-šá-tim 34a-ḫa-ab-bu-ub el-šú 35el-ki-šú-ma áš-ta-ka-an-šú 36a-na a-ḫi-ia 37um-mi dGiš mu-da-at [ka]-la-ma 38[iz-za-kàr-am a-na dGiš] 39[dGiš šá ta-mu-ru amêlu] 40[ta-ḫa-ab-bu-ub ki-ma áš-šá-tim el-šú] Col. II. 41áš-šum uš-[ta]-ma-ḫa-ru it-ti-ka 42dGiš šú-na-tam i-pa-šar 43dEn-ki-[dũ wa]-ši-ib ma-ḫar ḫa-ri-im-tim 44ur-[šá ir]-ḫa-mu di-da-šá(?) ip-tí-[e] 45[dEn-ki]-dũ im-ta-ši a-šar i-wa-al-du 46ûm, 6 ù 7 mu-ši-a-tim 47dEn-[ki-dũ] ti-bi-i-ma 48šá-[am-ka-ta] ir-ḫi 49ḫa-[ri-im-tum pa-a]-šá i-pu-šá-am-ma 50iz-za-[kàr-am] a-na dEn-ki-dũ 51a-na-tal-ka dEn-ki-dũ ki-ma ili ta-ba-áš-ši 52am-mi-nim it-ti na-ma-áš-te-e 53ta-at-ta-[na-al]-ak ṣi-ra-am[64] 54al-kam lu-úr-di-ka 55a-na libbi [Urukki] ri-bi-tim 56a-na bît [el]-lim mu-šá-bi šá A-nim 57dEn-ki-dũ ti-bi lu-ru-ka 58a-na Ê-[an]-na mu-šá-bi šá A-nim 59a-šar [dGiš gi]-it-ma-[lu] ne-pi-ši-tim 60ù at-[ta] ki-[ma Sal ta-ḫa]-bu-[ub]-šú 61ta-[ra-am-šú ki-ma] ra-ma-an-ka 62al-ka ti-ba i-[na] ga-ag-ga-ri 63ma-a-ag-ri-i-im 64iš-me a-wa-as-sa im-ta-ḫar ga-ba-šá 65mi-il-[kum] šá aššatim 66im-ta-ḳu-ut a-na libbi-šú 67iš-ḫu-ut li-ib-šá-am 68iš-ti-nam ú-la-ab-bi-iš-sú 69li-ib-[šá-am] šá-ni-a-am 70ši-i it-ta-al-ba-áš 71ṣa-ab-tat ga-as-su 72ki-ma [ili] i-ri-id-di-šú 73a-na gu-up-ri šá-ri-i-im 74a-šar tar-ba-ṣi-im 75i-na [áš]-ri-šú [im]-ḫu-ruri-ia-ú 76[ù šú-u dEn-ki-dũ i-lit-ta-šú šá-du-um-ma] 77[it-ti ṣabâti-ma ik-ka-la šam-ma] 78[it-ti bu-lim maš-ḳa-a i-šat-ti] 79[it-ti na-ma-áš-te-e mê i-ṭab lib-ba-šú] (Perhaps one additional line missing.) Col. III. 81ši-iz-ba šá na-ma-áš-te-e 82i-te-en-ni-ik 83a-ka-lam iš-ku-nu ma-ḫar-šú 84ib-tí-ik-ma i-na-at-tal 85ù ip-pa-al-la-as[65] 86ú-ul i-di dEn-ki-dũ 87aklam a-na a-ka-lim 88šikaram a-na šá-te-e-im 89la-a lum-mu-ud 90ḫa-ri-im-tum pi-šá i-pu-šá-am-ma 91iz-za-kàr-am a-na dEn-ki-dũ 92a-ku-ul ak-lam dEn-ki-dũ 93zi-ma-at ba-la-ṭi-im 94šikaram ši-ti ši-im-ti ma-ti 95i-ku-ul a-ak-lam dEn-ki-dũ 96a-di ši-bi-e-šú 97šikaram iš-ti-a-am 987 aṣ-ṣa-am-mi-im 99it-tap-šar kab-ta-tum i-na-an-gu 100i-li-iṣ libba-šú-ma 101pa-nu-šú [it]-tam-ru 102ul-tap-pi-it [lùŠÚ]-I 103šú-ḫu-ra-am pa-ga-ar-šú 104šá-am-nam ip-ta-šá-áš-ma 105a-we-li-iš i-we 106il-ba-áš li-ib-šá-am 107ki-ma mu-ti i-ba-áš-ši 108il-ki ka-ak-ka-šú 109la-bi ú-gi-ir-ri 110uš-sa-ak-pu re’ûti mu-ši-a-tim 111ut-tap-pi-iš šib-ba-ri 112la-bi uk-ta-ši-id 113it-ti-[lu] na-ki-[di-e] ra-bu-tum 114dEn-ki-dũ ma-aṣ-ṣa-ar-šú-nu 115a-we-lum giš-ru-um 116iš-te-en it-lum 117a-na [na-ki-di-e(?) i]-za-ak-ki-ir (About five lines missing.) Col. IV. (About eight lines missing.) 131i-ip-pu-uš ul-ṣa-am 132iš-ši-ma i-ni-i-šú 133i-ta-mar a-we-lam[66] 134iz-za-kàr-am a-na ḫarimtim 135šá-am-ka-at uk-ki-ši a-we-lam 136a-na mi-nim il-li-kam 137zi-ki-ir-šú lu-uš-šú 138ḫa-ri-im-tum iš-ta-si a-we-lam 139i-ba-uš-su-um-ma i-ta-mar-šú 140e-di-il e-eš ta-ḫi-[il-la]-am 141lim-nu a-la-ku ma-na-aḫ-[ti]-ka 142e-pi-šú i-pu-šá-am-ma 143iz-za-kàr-am a-na dEn-[ki-dũ] 144bi-ti-iš e-mu-tim ik …… 145ši-ma-a-at ni-ši-i-ma 146tu-a-(?)-ar e-lu-tim 147a-na âli(?) dup-šak-ki-i e-ṣi-en 148uk-la-at âli(?) e-mi-sa a-a-ḫa-tim 149a-na šarri šá Urukki ri-bi-tim 150pi-ti pu-uk epiši(-ši) a-na ḫa-a-a-ri 151a-na dGiš šarri šá Urukki ri-bi-tim 152pi-ti pu-uk epiši(-ši) 153a-na ḫa-a-a-ri 154áš-ša-at ši-ma-tim i-ra-aḫ-ḫi 155šú-ú pa-na-nu-um-ma 156mu-uk wa-ar-ka-nu 157i-na mi-il-ki šá ili ga-bi-ma 158i-na bi-ti-iḳ a-bu-un-na-ti-šú 159ši-ma-as-su 160a-na zi-ik-ri it-li-im 161i-ri-ku pa-nu-šú (About three lines missing.) [67] Col. V. (About six lines missing.) 171i-il-la-ak [dEn-ki-dũ i-na pa-ni] 172u-šá-am-ka-at [wa]-ar-ki-šú 173i-ru-ub-ma a-na libbi Urukki ri-bi-tim 174ip-ḫur um-ma-nu-um i-na ṣi-ri-šú 175iz-zi-za-am-ma i-na su-ḳi-im 176šá Urukki ri-bi-tim 177pa-aḫ-ra-a-ma ni-šú 178i-ta-wa-a i-na ṣi-ri-šú 179a-na ṣalam dGiš ma-ši-il pi-it-tam 180la-nam šá-pi-il 181si-ma …. [šá-ki-i pu]-uk-ku-ul 182............. i-pa-ka-du 183i-[na mâti da-an e-mu]-ki i-wa 184ši-iz-ba šá na-ma-aš-te-e 185i-te-en-ni-ik 186ka-a-a-na i-na [libbi] Urukki kak-ki-a-tum 187it-lu-tum ú-te-el-li-lu 188šá-ki-in ur-šá-nu 189a-na itli šá i-šá-ru zi-mu-šú 190a-na dGiš ki-ma i-li-im 191šá-ki-iš-šum me-iḫ-rù 192a-na dIš-ḫa-ra ma-a-a-lum 193na-di-i-ma 194dGiš it-[ti-il-ma wa-ar-ka-tim] 195i-na mu-ši in-ni-[ib-bi]-it 196i-na-ag-šá-am-ma 197it-ta-[zi-iz dEn-ki-dũ] i-na sûḳim 198ip-ta-ra-[aṣ a-la]-ak-tam 199šá dGiš 200[a-na e-pi-iš] da-na-ni-iš-šú (About three lines missing.) [68] Col. VI. (About four lines missing.) 208šar(?)-ḫa 209dGiš … 210i-na ṣi-ri-[šú il-li-ka-am dEn-ki-dũ] 211i-ḫa-an-ni-ib [pi-ir-ta-šú] 212it-bi-ma [il-li-ik] 213a-na pa-ni-šú 214it-tam-ḫa-ru i-na ri-bi-tum ma-ti 215dEn-ki-dũ ba-ba-am ip-ta-ri-ik 216i-na ši-pi-šú 217dGiš e-ri-ba-am ú-ul id-di-in 218iṣ-ṣa-ab-tu-ma ki-ma li-i-im 219i-lu-du 220zi-ip-pa-am ’i-bu-tu 221i-ga-rum ir-tu-tu 222dGiš ù dEn-ki-dũ 223iṣ-ṣa-ab-tu-ú-ma 224ki-ma li-i-im i-lu-du 225zi-ip-pa-am ’i-bu-tu 226i-ga-rum ir-tu-tú 227ik-mi-is-ma dGiš 228i-na ga-ag-ga-ri ši-ip-šú 229ip-ši-iḫ uz-za-šú-ma 230i-ni-iḫ i-ra-as-su 231iš-tu i-ra-su i-ni-ḫu 232dEn-ki-dũ a-na šá-ši-im 233iz-za-kàr-am a-na dGiš 234ki-ma iš-te-en-ma um-ma-ka 235ú-li-id-ka 236ri-im-tum šá su-pu-ri 237dNin-sun-na 238ul-lu e-li mu-ti ri-eš-ka 239šar-ru-tú šá ni-ši 240i-ši-im-kum dEn-lil 241 duppu 2 kam-ma 242šú-tu-ur e-li ………………… 243 4 šú-ši [62] Translation. Col. I. 1Gish sought to interpret the dream; 2Spoke to his mother: 3“My mother, during my night 4I became strong and moved about 5among the heroes; 6And from the starry heaven 7A meteor(?) of Anu fell upon me: 8I bore it and it grew heavy upon me, 9I became weak and its weight I could not endure. 10The land of Erech gathered about it. 11The heroes kissed its feet.1 12It was raised up before me. 13They stood me up.2 14I bore it and carried it to thee.” 15The mother of Gish, who knows all things, 16Spoke to Gish: 17“Some one, O Gish, who like thee 18In the field was born and 19Whom the mountain has reared, 20Thou wilt see (him) and [like a woman(?)] thou wilt rejoice. 21Heroes will kiss his feet. 22Thou wilt spare [him and wilt endeavor] 23To lead him to me.” 24He slept and saw another[63] 25Dream, which he reported to his mother: 26[“My mother,] I have seen another 27[Dream.] My likeness I have seen in the streets 28[Of Erech] of the plazas. 29An axe was brandished, and 30They gathered about him; 31And the axe made him angry. 32I saw him and I rejoiced, 33I loved him as a woman, 34I embraced him. 35I took him and regarded him 36As my brother.” 37The mother of Gish, who knows all things, 38[Spoke to Gish]: 39[“O Gish, the man whom thou sawest,] 40[Whom thou didst embrace like a woman]. Col II. 41(means) that he is to be associated with thee.” 42Gish understood the dream. 43[As] Enki[du] was sitting before the woman, 44[Her] loins(?) he embraced, her vagina(?) he opened. 45[Enkidu] forgot the place where he was born. 46Six days and seven nights 47Enkidu continued 48To cohabit with [the courtesan]. 49[The woman] opened her [mouth] and 50Spoke to Enkidu: 51“I gaze upon thee, O Enkidu, like a god art thou! 52Why with the cattle 53Dost thou [roam] across the field?[64] 54Come, let me lead thee 55into [Erech] of the plazas, 56to the holy house, the dwelling of Anu, 57O, Enkidu arise, let me conduct thee 58To Eanna, the dwelling of Anu, 59The place [where Gish is, perfect] in vitality. 60And thou [like a wife wilt embrace] him. 61Thou [wilt love him like] thyself. 62Come, arise from the ground 63(that is) cursed.” 64He heard her word and accepted her speech. 65The counsel of the woman 66Entered his heart. 67She stripped off a garment, 68Clothed him with one. 69Another garment 70She kept on herself. 71She took hold of his hand. 72Like [a god(?)] she brought him 73To the fertile meadow, 74The place of the sheepfolds. 75In that place they received food; 76[For he, Enkidu, whose birthplace was the mountain,] 77[With the gazelles he was accustomed to eat herbs,] 78[With the cattle to drink water,] 79[With the water beings he was happy.] (Perhaps one additional line missing.) Col. III. 81Milk of the cattle 82He was accustomed to suck. 83Food they placed before him, 84He broke (it) off and looked 85And gazed.[65] 86Enkidu had not known 87To eat food. 88To drink wine 89He had not been taught. 90The woman opened her mouth and 91Spoke to Enkidu: 92“Eat food, O Enkidu, 93The provender of life! 94Drink wine, the custom of the land!” 95Enkidu ate food 96Till he was satiated. 97Wine he drank, 98Seven goblets. 99His spirit was loosened, he became hilarious. 100His heart became glad and 101His face shone. 102[The barber(?)] removed 103The hair on his body. 104He was anointed with oil. 105He became manlike. 106He put on a garment, 107He was like a man. 108He took his weapon; 109Lions he attacked, 110(so that) the night shepherds could rest. 111He plunged the dagger; 112Lions he overcame. 113The great [shepherds] lay down; 114Enkidu was their protector. 115The strong man, 116The unique hero, 117To [the shepherds(?)] he speaks: (About five lines missing.) Col. IV. (About eight lines missing.) 131Making merry. 132He lifted up his eyes, 133He sees the man.[66] 134He spoke to the woman: 135“O, courtesan, lure on the man. 136Why has he come to me? 137His name I will destroy.” 138The woman called to the man 139Who approaches to him3 and he beholds him. 140“Away! why dost thou [quake(?)] 141Evil is the course of thy activity.”4 142Then he5 opened his mouth and 143Spoke to Enkidu: 144”[To have (?)] a family home 145Is the destiny of men, and 146The prerogative(?) of the nobles. 147For the city(?) load the workbaskets! 148Food supply for the city lay to one side! 149For the King of Erech of the plazas, 150Open the hymen(?), perform the marriage act! 151For Gish, the King of Erech of the plazas, 152Open the hymen(?), 153Perform the marriage act! 154With the legitimate wife one should cohabit. 155So before, 156As well as in the future.6 157By the decree pronounced by a god, 158From the cutting of his umbilical cord 159(Such) is his fate.” 160At the speech of the hero 161His face grew pale. (About three lines missing.) [67] Col. V. (About six lines missing.) 171[Enkidu] went [in front], 172And the courtesan behind him. 173He entered into Erech of the plazas. 174The people gathered about him. 175As he stood in the streets 176Of Erech of the plazas, 177The men gathered, 178Saying in regard to him: 179“Like the form of Gish he has suddenly become; 180shorter in stature. 181[In his structure high(?)], powerful, 182.......... overseeing(?) 183In the land strong of power has he become. 184Milk of cattle 185He was accustomed to suck.” 186Steadily(?) in Erech ..... 187The heroes rejoiced. 188He became a leader. 189To the hero of fine appearance, 190To Gish, like a god, 191He became a rival to him.7 192For Ishḫara a couch 193Was stretched, and 194Gish [lay down, and afterwards(?)] 195In the night he fled. 196He approaches and 197[Enkidu stood] in the streets. 198He blocked the path 199of Gish. 200At the exhibit of his power, (About three lines missing.) [68] Col. VI. (About four lines missing.) 208Strong(?) … 209Gish 210Against him [Enkidu proceeded], 211[His hair] luxuriant. 212He started [to go] 213Towards him. 214They met in the plaza of the district. 215Enkidu blocked the gate 216With his foot, 217Not permitting Gish to enter. 218They seized (each other), like oxen, 219They fought. 220The threshold they demolished; 221The wall they impaired. 222Gish and Enkidu 223Seized (each other). 224Like oxen they fought. 225The threshold they demolished; 226The wall they impaired. 227Gish bent 228His foot to the ground,8 229His wrath was appeased, 230His breast was quieted. 231When his breast was quieted, 232Enkidu to him 233Spoke, to Gish: 234“As a unique one, thy mother 235bore thee. 236The wild cow of the stall,9 237Ninsun, 238Has exalted thy head above men. 239Kingship over men 240Enlil has decreed for thee. 241Second tablet, 242enlarged beyond [the original(?)]. 243240 lines. [69] 1 I.e., paid homage to the meteor. 2 I.e., the heroes of Erech raised me to my feet, or perhaps in the sense of “supported me.” 3 I.e., Enkidu. 4 I.e., “thy way of life.” 5 I.e., the man. 6 I.e., an idiomatic phrase meaning “for all times.” 7 I.e., Enkidu became like Gish, godlike. Cf. col. 2, 11. 8 He was thrown and therefore vanquished. 9 Epithet given to Ninsun. See the commentary to the line. Commentary on the Pennsylvania Tablet. Line 1. The verb tibû with pašâru expresses the aim of Gish to secure an interpretation for his dream. This disposes of Langdon’s note 1 on page 211 of his edition, in which he also erroneously speaks of our text as “late.” Pašâru is not a variant of zakâru. Both verbs occur just as here in the Assyrian version I, 5, 25. Line 3. ina šât mušitia, “in this my night,” i.e., in the course of this night of mine. A curious way of putting it, but the expression occurs also in the Assyrian version, e.g., I, 5, 26 (parallel passage to ours) and II, 4a, 14. In the Yale tablet we find, similarly, mu-ši-it-ka (l. 262), “thy night,” i.e., “at night to thee.” Line 5. Before Langdon put down the strange statement of Gish “wandering about in the midst of omens” (misreading id-da-tim for it-lu-tim), he might have asked himself the question, what it could possibly mean. How can one walk among omens? Line 6. ka-ka-bu šá-ma-i must be taken as a compound term for “starry heaven.” The parallel passage in the Assyrian version (Tablet I, 5, 27) has the ideograph for star, with the plural sign as a variant. Literally, therefore, “The starry heaven (or “the stars in heaven”) was there,” etc. Langdon’s note 2 on page 211 rests on an erroneous reading. Line 7. kiṣru šá Anim, “mass of Anu,” appears to be the designation of a meteor, which might well be described as a “mass” coming from Anu, i.e., from the god of heaven who becomes the personification of the heavens in general. In the Assyrian version (I, 5, 28) we have kima ki-iṣ-rù, i.e., “something like a mass of heaven.” Note also I, 3, 16, where in a description of Gilgamesh, his strength is said to be “strong like a mass (i.e., a meteor) of heaven.” Line 9. For nuššašu ûl iltê we have a parallel in the Hebrew phrase נלְַפָסֵתִי נשַׂפָס (Isaiah 1, 14). Line 10. Uruk mâtum, as the designation for the district of Erech, occurs in the Assyrian version, e.g., I, 5, 31, and IV, 2, 38; also to be supplied, I, 6, 23. For paḫir the parallel in the Assyrian version has iz-za-az (I, 5, 31), but VI, 197, we find paḫ-ru and paḫ-ra. Line 17. mi-in-di does not mean “truly” as Langdon translates, but “some one.” It occurs also in the Assyrian version X, 1, 13, mi-in-di-e ma-an-nu-ṵ, “this is some one who,” etc. [70] Line 18. Cf. Assyrian version I, 5, 3, and IV, 4, 7, ina ṣiri âlid—both passages referring to Enkidu. Line 21. Cf. Assyrian version II, 3b, 38, with malkê, “kings,” as a synonym of itlutum. Line 23. ta-tar-ra-as-sú from tarâṣu, “direct,” “guide,” etc. Line 24. I take uš-ti-nim-ma as III, 2, from išênu (יָשֵׁן), the verb underlying šittu, “sleep,” and šuttu, “dream.” Line 26. Cf. Assyrian version I, 6, 21—a complete parallel. Line 28. Uruk ri-bi-tim, the standing phrase in both tablets of the old Babylonian version, for which in the Assyrian version we have Uruk su-pu-ri. The former term suggests the “broad space” outside of the city or the “common” in a village community, while supûri, “enclosed,” would refer to the city within the walls. Dr. W. F. Albright (in a private communication) suggests “Erech of the plazas” as a suitable translation for Uruk ribîtim. A third term, Uruk mâtum (see above, note to line 10), though designating rather the district of which Erech was the capital, appears to be used as a synonym to Uruk ribîtim, as may be concluded from the phrase i-na ri-bi-tum ma-ti (l. 214 of the Pennsylvania tablet), which clearly means the “plaza” of the city. One naturally thinks of רְחֹבֹת עִיר in Genesis 10, 11—the equivalent of Babylonian ri-bi-tu âli—which can hardly be the name of a city. It appears to be a gloss, as is הִיַפָס הָעִיּר הַגְּדֹלָה at the end of v. 12. The latter gloss is misplaced, since it clearly describes “Nineveh,” mentioned in v. 11. Inasmuch as רְחֹבֹת עִיר immediately follows the mention of Nineveh, it seems simplest to take the phrase as designating the “outside” or “suburbs” of the city, a complete parallel, therefore, to ri-bi-tu mâti in our text. Nineveh, together with the “suburbs,” forms the “great city.” Uruk ribîtim is, therefore, a designation for “greater Erech,” proper to a capital city, which by its gradual growth would take in more than its original confines. “Erech of the plazas” must have come to be used as a honorific designation of this important center as early as 2000 B. C., whereas later, perhaps because of its decline, the epithet no longer seemed appropriate and was replaced by the more modest designation of “walled Erech,” with an allusion to the tradition which ascribed the building of the wall of the city to Gilgamesh. At all [71]events, all three expressions, “Erech of the plazas,” “Erech walled” and “Erech land,” are to be regarded as synonymous. The position once held by Erech follows also from its ideographic designation (Brünnow No. 4796) by the sign “house” with a “gunufied” extension, which conveys the idea of Unu = šubtu, or “dwelling” par excellence. The pronunciation Unug or Unuk (see the gloss u-nu-uk, VR 23, 8a), composed of unu, “dwelling,” and ki, “place,” is hardly to be regarded as older than Uruk, which is to be resolved into uru, “city,” and ki, “place,” but rather as a play upon the name, both Unu + ki and Uru + ki conveying the same idea of the city or the dwelling place par excellence. As the seat of the second oldest dynasty according to Babylonian traditions (see Poebel’s list in Historical and Grammatical Texts No. 2), Erech no doubt was regarded as having been at one time “the city,” i.e., the capital of the entire Euphrates Valley. Line 31. A difficult line for which Langdon proposes the translation: “Another axe seemed his visage”!!—which may be picturesque, but hardly a description befitting a hero. How can a man’s face seem to be an axe? Langdon attaches šá-ni in the sense of “second” to the preceding word “axe,” whereas šanî bunušu, “change of his countenance” or “his countenance being changed,” is to be taken as a phrase to convey the idea of “being disturbed,” “displeased” or “angry.” The phrase is of the same kind as the well-known šunnu ṭêmu, “changing of reason,” to denote “insanity.” See the passages in Muss-Arnolt, Assyrian Dictionary, pp. 355 and 1068. In Hebrew, too, we have the same two phrases, e.g., וַיְשַׁנֹּו ַפָסֶת־טַעְמֹו (I Sam. 21, 14 = Ps. 34, 1), “and he changed his reason,” i.e., feigned insanity and מְשַׁנֶּה פָּנָיו (Job 14, 20), “changing his face,” to indicate a radical alteration in the frame of mind. There is a still closer parallel in Biblical Aramaic: Dan. 3, 19, “The form of his visage was changed,” meaning “he was enraged.” Fortunately, the same phrase occurs also in the Yale tablet (l. 192), šá-nu-ú bu-nu-šú, in a connection which leaves no doubt that the aroused fury of the tyrant Ḫuwawa is described by it: ”Ḫuwawa heard and his face was changed” precisely, therefore, as we should say—following Biblical usage—“his countenance fell.” Cf. also the phrase pânušu arpu, “his countenance [72]was darkened” (Assyrian version I, 2, 48), to express “anger.” The line, therefore, in the Pennsylvania tablet must describe Enkidu’s anger. With the brandishing of the axe the hero’s anger was also stirred up. The touch was added to prepare us for the continuation in which Gish describes how, despite this (or perhaps just because of it), Enkidu seemed so attractive that Gish instantly fell in love with him. May perhaps the emphatic form ḫaṣinumma (line 31) against ḫaṣinu (line 29) have been used to indicate “The axe it was,” or “because of the axe?” It would be worth while to examine other texts of the Hammurabi period with a view of determining the scope in the use and meaning of the emphatic ma when added to a substantive. Line 32. The combination amur ù aḫtadu occurs also in the El-Amarna Letters, No. 18, 12. Line 34. In view of the common Hebrew, Syriac and Arabic חָבַב “to love,” it seems preferable to read here, as in the other passages in the Assyrian versions (I, 4, 15; 4, 35; 6, 27, etc.), a-ḫa-ab-bu-ub, aḫ-bu-ub, iḫ-bu-bu, etc. (instead of with p), and to render “embrace.” Lines 38–40, completing the column, may be supplied from the Assyrian version I, 6, 30–32, in conjunction with lines 33–34 of our text. The beginning of line 32 in Jensen’s version is therefore to be filled out [ta-ra-am-šú ki]-i. Line 43. The restoration at the beginning of this line En-ki-[dũ wa]-ši-ib ma-ḫar ḫa-ri-im-tim enables us to restore also the beginning of the second tablet of the Assyrian version (cf. the colophon of the fragment 81, 7–27, 93, in Jeremias, Izdubar-Nimrod, plate IV = Jensen, p. 134), [dEn-ki-dũ wa-ši-ib] ma-ḫar-šá. Line 44. The restoration of this line is largely conjectural, based on the supposition that its contents correspond in a general way to I, 4, 16, of the Assyrian version. The reading di-da is quite certain, as is also ip-ti-[e]; and since both words occur in the line of the Assyrian version in question, it is tempting to supply at the beginning ur-[šá] = “her loins” (cf. Holma, Namen der Körperteile, etc., p. 101), which is likewise found in the same line of the Assyrian version. At all events the line describes the fascination exercised [73]upon Enkidu by the woman’s bodily charms, which make him forget everything else. Lines 46–47 form a parallel to I, 4, 21, of the Assyrian version. The form šamkatu, “courtesan,” is constant in the old Babylonian version (ll. 135 and 172), as against šamḫatu in the Assyrian version (I, 3, 19, 40, 45; 4, 16), which also uses the plural šam-ḫa-a-ti (II, 3b, 40). The interchange between ḫ and k is not without precedent (cf. Meissner, Altbabylonisches Privatrecht, page 107, note 2, and more particularly Chiera, List of Personal Names, page 37). In view of the evidence, set forth in the Introduction, for the assumption that the Enkidu story has been combined with a tale of the evolution of primitive man to civilized life, it is reasonable to suggest that in the original Enkidu story the female companion was called šamkatu, “courtesan,” whereas in the tale of the primitive man, which was transferred to Enkidu, the associate was ḫarimtu, a “woman,” just as in the Genesis tale, the companion of Adam is simply called ishshâ, “woman.” Note that in the Assyrian parallel (Tablet I, 4, 26) we have two readings, ir-ḫi (imperf.) and a variant i-ri-ḫi (present). The former is the better reading, as our tablet shows. Lines 49–59 run parallel to the Assyrian version I, 4, 33–38, with slight variations which have been discussed above, p. 58, and from which we may conclude that the Assyrian version represents an independent redaction. Since in our tablet we have presumably the repetition of what may have been in part at least set forth in the first tablet of the old Babylonian version, we must not press the parallelism with the first tablet of the Assyrian version too far; but it is noticeable nevertheless (1) that our tablet contains lines 57–58 which are not represented in the Assyrian version, and (2) that the second speech of the “woman” beginning, line 62, with al-ka, “come” (just as the first speech, line 54), is likewise not found in the first tablet of the Assyrian version; which on the other hand contains a line (39) not in the Babylonian version, besides the detailed answer of Enkidu (I 4, 42–5, 5). Line 6, which reads “Enkidu and the woman went (il-li-ku) to walled Erech,” is also not found in the second tablet of the old Babylonian version. Line 63. For magrû, “accursed,” see the frequent use in Astrological texts (Jastrow, Religion Babyloniens und Assyriens II, page [74]450, note 2). Langdon, by his strange error in separating ma-a-ag-ri-im into two words ma-a-ak and ri-i-im, with a still stranger rendering: “unto the place yonder of the shepherds!!”, naturally misses the point of this important speech. Line 64 corresponds to I, 4, 40, of the Assyrian version, which has an additional line, leading to the answer of Enkidu. From here on, our tablet furnishes material not represented in the Assyrian version, but which was no doubt included in the second tablet of that version of which we have only a few fragments. Line 70 must be interpreted as indicating that the woman kept one garment for herself. Ittalbaš would accordingly mean, “she kept on.” The female dress appears to have consisted of an upper and a lower garment. Line 72. The restoration “like a god” is favored by line 51, where Enkidu is likened to a god, and is further confirmed by l. 190. Line 73. gupru is identical with gu-up-ri (Thompson, Reports of the Magicians and Astrologers, etc., 223 rev. 2 and 223a rev. 8), and must be correlated to gipâru (Muss-Arnolt, Assyrian Dictionary, p. 229a), “planted field,” “meadow,” and the like. Thompson’s translation “men” (as though a synonym of gabru) is to be corrected accordingly. Line 74. There is nothing missing between a-šar and tar-ba-ṣi-im. Line 75. ri-ia-ú, which Langdon renders “shepherd,” is the equivalent of the Arabic riʿy and Hebrew רְעִי “pasturage,” “fodder.” We have usually the feminine form ri-i-tu (Muss-Arnolt, Assyrian Dictionary, p. 990b). The break at the end of the second column is not serious. Evidently Enkidu, still accustomed to live like an animal, is first led to the sheepfolds, and this suggests a repetition of the description of his former life. Of the four or five lines missing, we may conjecturally restore four, on the basis of the Assyrian version, Tablet I, 4, 2–5, or I, 2, 39–41. This would then join on well to the beginning of column 3. Line 81. Both here and in l. 52 our text has na-ma-áš-te-e, as against nam-maš-ši-i in the Assyrian version, e.g., Tablet I, 2, 41; 4, 5, etc.,—the feminine form, therefore, as against the masculine. Langdon’s note 3 on page 213 is misleading. In astrological texts we also find nam-maš-te; e.g., Thompson, Reports of the Magicians and Astrologers, etc., No. 200, Obv. 2. [75] Line 93. zi-ma-at (for simat) ba-la-ṭi-im is not “conformity of life” as Langdon renders, but that which “belongs to life” like si-mat pag-ri-šá, “belonging to her body,” in the Assyrian version III, 2a, 3 (Jensen, page 146). “Food,” says the woman, “is the staff of life.” Line 94. Langdon’s strange rendering “of the conditions and fate of the land” rests upon an erroneous reading (see the corrections, Appendix I), which is the more inexcusable because in line 97 the same ideogram, Kàš = šikaru, “wine,” occurs, and is correctly rendered by him. Šimti mâti is not the “fate of the land,” but the “fixed custom of the land.” Line 98. aṣ-ṣa-mi-im (plural of aṣṣamu), which Langdon takes as an adverb in the sense of “times,” is a well-known word for a large “goblet,” which occurs in Incantation texts, e.g., CT XVI, 24, obv. 1, 19, mê a-ṣa-am-mi-e šú-puk, “pour out goblets of water.” Line 18 of the passage shoves that aṣammu is a Sumerian loan word. Line 99. it-tap-šar, I, 2, from pašâru, “loosen.” In combination with kabtatum (from kabitatum, yielding two forms: kabtatum, by elision of i, and kabittu, by elision of a), “liver,” pašâru has the force of becoming cheerful. Cf. ka-bit-ta-ki lip-pa-šir (ZA V., p. 67, line 14). Line 100, note the customary combination of “liver” (kabtatum) and “heart” (libbu) for “disposition” and “mind,” just as in the standing phrase in penitential prayers: “May thy liver be appeased, thy heart be quieted.” Line 102. The restoration [lùŠÚ]-I = gallabu “barber” (Delitzsch, Sumer. Glossar, p. 267) was suggested to me by Dr. H. F. Lutz. The ideographic writing “raising the hand” is interesting as recalling the gesture of shaving or cutting. Cf. a reference to a barber in Lutz, Early Babylonian Letters from Larsa, No. 109, 6. Line 103. Langdon has correctly rendered šuḫuru as “hair,” and has seen that we have here a loan-word from the Sumerian Suḫur = kimmatu, “hair,” according to the Syllabary Sb 357 (cf. Delitzsch, Sumer. Glossar., p. 253). For kimmatu, “hair,” more specifically hair of the head and face, see Holma, Namen der Körperteile, page 3. The same sign Suḫur or Suḫ (Brünnow No. 8615), with Lal, i.e., “hanging hair,” designates the “beard” (ziḳnu, cf. Brünnow, No. 8620, and Holma, l. c., p. 36), and it is interesting to [76]note that we have šuḫuru (introduced as a loan-word) for the barbershop, according to II R, 21, 27c (= CT XII, 41). Ê suḫur(ra) (i.e., house of the hair) = šú-ḫu-ru. In view of all this, we may regard as assured Holma’s conjecture to read šú-[ḫur-ma-šú] in the list 93074 obv. (MVAG 1904, p. 203; and Holma, Beiträge z. Assyr. Lexikon, p. 36), as the Akkadian equivalent to Suḫur-Maš-Ḫa and the name of a fish, so called because it appeared to have a double “beard” (cf. Holma, Namen der Körperteile). One is tempted, furthermore, to see in the difficult word שכירה (Isaiah 7, 20) a loan-word from our šuḫuru, and to take the words ַפָסֶת־הָרַֹפָסשׁ וְשַׂעַר הָרַגְלַיִם “the head and hair of the feet” (euphemistic for the hair around the privates), as an explanatory gloss to the rare word שכירה for “hair” of the body in general—just as in the passage in the Pennsylvania tablet. The verse in Isaiah would then read, “The Lord on that day will shave with the razor the hair (השכירה), and even the beard will be removed.” The rest of the verse would represent a series of explanatory glosses: (a) “Beyond the river” (i.e., Assyria), a gloss to יְגַלַּח (b) “with the king of Assyria,” a gloss to בְּתַעַר “with a razor;” and (c) “the hair of the head and hair of the feet,” a gloss to השכירה. For “hair of the feet” we have an interesting equivalent in Babylonian šu-ḫur (and šú-ḫu-ur) šêpi (CT XII, 41, 23–24 c-d). Cf. also Boissier, Documents Assyriens relatifs aux Présages, p. 258, 4–5. The Babylonian phrase is like the Hebrew one to be interpreted as a euphemism for the hair around the male or female organ. To be sure, the change from ה to כ in השכירה constitutes an objection, but not a serious one in the case of a loan-word, which would aim to give the pronunciation of the original word, rather than the correct etymological equivalent. The writing with aspirated כ fulfills this condition. (Cf. šamkatum and šamḫatum, above p. 73). The passage in Isaiah being a reference to Assyria, the prophet might be tempted to use a foreign word to make his point more emphatic. To take השכירה as “hired,” as has hitherto been done, and to translate “with a hired razor,” is not only to suppose a very wooden metaphor, but is grammatically difficult, since השכירח would be a feminine adjective attached to a masculine substantive. Coming back to our passage in the Pennsylvania tablet, it is to [77]be noted that Enkidu is described as covered “all over his body with hair” (Assyrian version, Tablet I, 2, 36) like an animal. To convert him into a civilized man, the hair is removed. Line 107. mutu does not mean “husband” here, as Langdon supposes, but must be taken as in l. 238 in the more general sense of “man,” for which there is good evidence. Line 109. la-bi (plural form) are “lions”—not “panthers” as Langdon has it. The verb ú-gi-ir-ri is from gâru, “to attack.” Langdon by separating ú from gi-ir-ri gets a totally wrong and indeed absurd meaning. See the corrections in the Appendix. He takes the sign ú for the copula (!!) which of course is impossible. Line 110. Read uš-sa-ak-pu, III, 1, of sakâpu, which is frequently used for “lying down” and is in fact a synonym of ṣalâlu. See Muss-Arnolt, Assyrian Dictionary, page 758a. The original has very clearly Síb (= rê’u, “shepherd”) with the plural sign. The “shepherds of the night,” who could now rest since Enkidu had killed the lions, are of course the shepherds who were accustomed to watch the flocks during the night. Line 111. ut-tap-pi-iš is II, 2, napâšu, “to make a hole,” hence “to plunge” in connection with a weapon. Šib-ba-ri is, of course, not “mountain goats,” as Langdon renders, but a by-form to šibbiru, “stick,” and designates some special weapon. Since on seal cylinders depicting Enkidu killing lions and other animals the hero is armed with a dagger, this is presumably the weapon šibbaru. Line 113. Langdon’s translation is again out of the question and purely fanciful. The traces favor the restoration na-ki-[di-e], “shepherds,” and since the line appears to be a parallel to line 110, I venture to suggest at the beginning [it-ti]-lu from na’âlu, “lie down”—a synonym, therefore, to sakâpu in line 110. The shepherds can sleep quietly after Enkidu has become the “guardian” of the flocks. In the Assyrian version (tablet II, 3a, 4) Enkidu is called a na-kid, “shepherd,” and in the preceding line we likewise have lùNa-Kid with the plural sign, i.e., “shepherds.” This would point to nakidu being a Sumerian loan-word, unless it is vice versa, a word that has gone over into the Sumerian from Akkadian. Is perhaps the fragment in question (K 8574) in the Assyrian version (Haupt’s ed. No. 25) the parallel to our passage? If in line 4 of this fragment we could read šú for sa, i.e., na-kid-šú-nu, “their shepherd, we would have a [78]parallel to line 114 of the Pennsylvania tablet, with na-kid as a synonym to maṣṣaru, “protector.” The preceding line would then be completed as follows: [it-ti-lu]-nim-ma na-kidmeš [ra-bu-tum] (or perhaps only it-ti-lu-ma, since the nim is not certain) and would correspond to line 113 of the Pennsylvania tablet. Inasmuch as the writing on the tiny fragment is very much blurred, it is quite possible that in line 2 we must read šib-ba-ri (instead of bar-ba-ri), which would furnish a parallel to line 111 of the Pennsylvania tablet. The difference between Bar and Šib is slight, and the one sign might easily be mistaken for the other in the case of close writing. The continuation of line 2 of the fragment would then correspond to line 112 of the Pennsylvania tablet, while line 1 of the fragment might be completed [re-e]-u-ti(?) šá [mu-ši-a-tim], though this is by no means certain. The break at the close of column 3 (about 5 lines) and the top of column 4 (about 8 lines) is a most serious interruption in the narrative, and makes it difficult to pick up the thread where the tablet again becomes readable. We cannot be certain whether the “strong man, the unique hero” who addresses some one (lines 115–117) is Enkidu or Gish or some other personage, but presumably Gish is meant. In the Assyrian version, Tablet I, 3, 2 and 29, we find Gilgamesh described as the “unique hero” and in l. 234 of the Pennsylvania tablet Gish is called “unique,” while again, in the Assyrian version, Tablet I, 2, 15 and 26, he is designated as gašru as in our text. Assuming this, whom does he address? Perhaps the shepherds? In either case he receives an answer that rejoices him. If the fragment of the Assyrian version (K 8574) above discussed is the equivalent to the close of column 3 of the Pennsylvania tablet, we may go one step further, and with some measure of assurance assume that Gish is told of Enkidu’s exploits and that the latter is approaching Erech. This pleases Gish, but Enkidu when he sees Gish(?) is stirred to anger and wants to annihilate him. At this point, the “man” (who is probably Gish, though the possibility of a third personage must be admitted) intervenes and in a long speech sets forth the destiny and higher aims of mankind. The contrast between Enkidu and Gish (or the third party) is that between the primitive [79]savage and the civilized being. The contrast is put in the form of an opposition between the two. The primitive man is the stronger and wishes to destroy the one whom he regards as a natural foe and rival. On the other hand, the one who stands on a higher plane wants to lift his fellow up. The whole of column 4, therefore, forms part of the lesson attached to the story of Enkidu, who, identified with man in a primitive stage, is made the medium of illustrating how the higher plane is reached through the guiding influences of the woman’s hold on man, an influence exercised, to be sure, with the help of her bodily charms. Line 135. uk-ki-ši (imperative form) does not mean “take away,” as Langdon (who entirely misses the point of the whole passage) renders, but on the contrary, “lure him on,” “entrap him,” and the like. The verb occurs also in the Yale tablet, ll. 183 and 186. Line 137. Langdon’s note to lu-uš-šú had better be passed over in silence. The form is II. 1, from ešû, “destroy.” Line 139. Since the man whom the woman calls approaches Enkidu, the subject of both verbs is the man, and the object is Enkidu; i.e., therefore, “The man approaches Enkidu and beholds him.” Line 140. Langdon’s interpretation of this line again is purely fanciful. E-di-il cannot, of course, be a “phonetic variant” of edir; and certainly the line does not describe the state of mind of the woman. Lines 140–141 are to be taken as an expression of amazement at Enkidu’s appearance. The first word appears to be an imperative in the sense of “Be off,” “Away,” from dâlu, “move, roam.” The second word e-eš, “why,” occurs with the same verb dâlu in the Meissner fragment: e-eš ta-da-al (column 3, 1), “why dost thou roam about?” The verb at the end of the line may perhaps be completed to ta-ḫi-il-la-am. The last sign appears to be am, but may be ma, in which case we should have to complete simply ta-ḫi-il-ma. Taḫîl would be the second person present of ḫîlu. Cf. i-ḫi-il, frequently in astrological texts, e.g., Virolleaud, Adad No. 3, lines 21 and 33. Line 141. The reading lim-nu at the beginning, instead of Langdon’s mi-nu, is quite certain, as is also ma-na-aḫ-ti-ka instead of what Langdon proposes, which gives no sense whatever. Manaḫtu in the sense of the “toil” and “activity of life” (like עָמָל throughout the Book of Ecclesiastes) occurs in the introductory lines to [80]the Assyrian version of the Epic I, 1, 8, ka-lu ma-na-aḫ-ti-[šu], “all of his toil,” i.e., all of his career. Line 142. The subject of the verb cannot be the woman, as Langdon supposes, for the text in that case, e.g., line 49, would have said pi-šá (“her mouth”) not pi-šú (“his mouth”). The long speech, detailing the function and destiny of civilized man, is placed in the mouth of the man who meets Enkidu. In the Introduction it has been pointed out that lines 149 and 151 of the speech appear to be due to later modifications of the speech designed to connect the episode with Gish. Assuming this to be the case, the speech sets forth the following five distinct aims of human life: (1) establishing a home (line 144), (2) work (line 147), (3) storing up resources (line 148), (4) marriage (line 150), (5) monogamy (line 154); all of which is put down as established for all time by divine decree (lines 155–157), and as man’s fate from his birth (lines 158–159). Line 144. bi-ti-iš e-mu-ti is for bîti šá e-mu-ti, just as ḳab-lu-uš Ti-a-ma-ti (Assyrian Creation Myth, IV, 65) stands for ḳablu šá Tiamti. Cf. bît e-mu-ti (Assyrian version, IV, 2, 46 and 48). The end of the line is lost beyond recovery, but the general sense is clear. Line 146. tu-a-ar is a possible reading. It may be the construct of tu-a-ru, of frequent occurrence in legal texts and having some such meaning as “right,” “claim” or “prerogative.” See the passages given by Muss-Arnolt, Assyrian Dictionary, p. 1139b. Line 148. The reading uk-la-at, “food,” and then in the wider sense “food supply,” “provisions,” is quite certain. The fourth sign looks like the one for “city.” E-mi-sa may stand for e-mid-sa, “place it.” The general sense of the line, at all events, is clear, as giving the advice to gather resources. It fits in with the Babylonian outlook on life to regard work and wealth as the fruits of work and as a proper purpose in life. Line 150 (repeated lines 152–153) is a puzzling line. To render piti pûk epši (or epiši), as Langdon proposes, “open, addressing thy speech,” is philologically and in every other respect inadmissible. The word pu-uk (which Langdon takes for “thy mouth”!!) can, of course, be nothing but the construct form of pukku, which occurs in the Assyrian version in the sense of “net” (pu-uk-ku I, 2, 9 and 21, and also in the colophon to the eleventh tablet furnishing the [81]beginning of the twelfth tablet (Haupt’s edition No. 56), as well as in column 2, 29, and column 3, 6, of this twelfth tablet). In the two last named passages pukku is a synonym of mekû, which from the general meaning of “enclosure” comes to be a euphemistic expression for the female organ. So, for example, in the Assyrian Creation Myth, Tablet IV, 66 (synonym of ḳablu, “waist,” etc.). See Holma, Namen der Körperteile, page 158. Our word pukku must be taken in this same sense as a designation of the female organ—perhaps more specifically the “hymen” as the “net,” though the womb in general might also be designated as a “net” or “enclosure.” Kak-(ši) is no doubt to be read epši, as Langdon correctly saw; or perhaps better, epiši. An expression like ip-ši-šú lul-la-a (Assyrian version, I, 4, 13; also line 19, i-pu-us-su-ma lul-la-a), with the explanation šipir zinništi, “the work of woman” (i.e., after the fashion of woman), shows that epêšu is used in connection with the sexual act. The phrase pitî pûk epiši a-na ḫa-a-a-ri, literally “open the net, perform the act for marriage,” therefore designates the fulfillment of the marriage act, and the line is intended to point to marriage with the accompanying sexual intercourse as one of the duties of man. While the general meaning is thus clear, the introduction of Gish is puzzling, except on the supposition that lines 149 and 151 represent later additions to connect the speech, detailing the advance to civilized life, with the hero. See above, p. 45 seq. Line 154. aššat šimâtim is the “legitimate wife,” and the line inculcates monogamy as against promiscuous sexual intercourse. We know that monogamy was the rule in Babylonia, though a man could in addition to the wife recognized as the legalized spouse take a concubine, or his wife could give her husband a slave as a concubine. Even in that case, according to the Hammurabi Code, §§145–146, the wife retained her status. The Code throughout assumes that a man has only one wife—the aššat šimâtim of our text. The phrase “so” (or “that”) before “as afterwards” is to be taken as an idiomatic expression—“so it was and so it should be for all times”—somewhat like the phrase maḫriam ù arkiam, “for all times,” in legal documents (CT VIII, 38c, 22–23). For the use of mûk see Behrens, Assyrisch-Babylonische Briefe, p. 3. Line 158. i-na bi-ti-iḳ a-bu-un-na-ti-šú. Another puzzling line, for which Langdon proposes “in the work of his presence,” which [82]is as obscure as the original. In a note he says that apunnâti means “nostrils,” which is certainly wrong. There has been considerable discussion about this term (see Holma, Namen der Körperteile, pages 150 and 157), the meaning of which has been advanced by Christian’s discussion in OLZ 1914, p. 397. From this it appears that it must designate a part of the body which could acquire a wider significance so as to be used as a synonym for “totality,” since it appears in a list of equivalent for Dur = nap-ḫa-ru, “totality,” ka-lu-ma, “all,” a-bu-un-na-tum e-ṣi-im-tum, “bony structure,” and kul-la-tum, “totality” (CT XII, 10, 7–10). Christian shows that it may be the “navel,” which could well acquire a wider significance for the body in general; but we may go a step further and specify the “umbilical cord” (tentatively suggested also by Christian) as the primary meaning, then the “navel,” and from this the “body” in general. The structure of the umbilical cord as a series of strands would account for designating it by a plural form abunnâti, as also for the fact that one could speak of a right and left side of the appunnâti. To distinguish between the “umbilical cord” and the “navel,” the ideograph Dur (the common meaning of which is riksu, “bond” [Delitzsch, Sumer. Glossar., p. 150]), was used for the former, while for the latter Li Dur was employed, though the reading in Akkadian in both cases was the same. The expression “with (or at) the cutting of his umbilical cord” would mean, therefore, “from his birth”—since the cutting of the cord which united the child with the mother marks the beginning of the separate life. Lines 158–159, therefore, in concluding the address to Enkidu, emphasize in a picturesque way that what has been set forth is man’s fate for which he has been destined from birth. [See now Albright’s remarks on abunnatu in the Revue d’Assyriologie 16, pp. 173–175, with whose conclusion, however, that it means primarily “backbone” and then “stature,” I cannot agree.] In the break of about three lines at the bottom of column 4, and of about six at the beginning of column 5, there must have been set forth the effect of the address on Enkidu and the indication of his readiness to accept the advice; as in a former passage (line 64), Enkidu showed himself willing to follow the woman. At all events the two now proceed to the heart of the city. Enkidu is in front [83]and the woman behind him. The scene up to this point must have taken place outside of Erech—in the suburbs or approaches to the city, where the meadows and the sheepfolds were situated. Line 174. um-ma-nu-um are not the “artisans,” as Langdon supposes, but the “people” of Erech, just as in the Assyrian version, Tablet IV, 1, 40, where the word occurs in connection with i-dip-pi-ir, which is perhaps to be taken as a synonym of paḫâru, “gather;” so also i-dip-pir (Tablet I, 2, 40) “gathers with the flock.” Lines 180–182 must have contained the description of Enkidu’s resemblance to Gish, but the lines are too mutilated to permit of any certain restoration. See the corrections (Appendix) for a suggested reading for the end of line 181. Line 183 can be restored with considerable probability on the basis of the Assyrian version, Tablet I, 3, 3 and 30, where Enkidu is described as one “whose power is strong in the land.” Lines 186–187. The puzzling word, to be read apparently kak-ki-a-tum, can hardly mean “weapons,” as Langdon proposes. In that case we should expect kakkê; and, moreover, to so render gives no sense, especially since the verb ú-te-el-li-lu is without much question to be rendered “rejoiced,” and not “purified.” Kakkiatum—if this be the correct reading—may be a designation of Erech like ribîtim. Lines 188–189 are again entirely misunderstood by Langdon, owing to erroneous readings. See the corrections in the Appendix. Line 190. i-li-im in this line is used like Hebrew Elohîm, “God.” Line 191. šakiššum = šakin-šum, as correctly explained by Langdon. Line 192. With this line a new episode begins which, owing to the gap at the beginning of column 6, is somewhat obscure. The episode leads to the hostile encounter between Gish and Enkidu. It is referred to in column 2 of the fourth tablet of the Assyrian version. Lines 35–50—all that is preserved of this column—form in part a parallel to columns 5–6 of the Pennsylvania tablet, but in much briefer form, since what on the Pennsylvania tablet is the incident itself is on the fourth tablet of the Assyrian version merely a repeated summary of the relationship between the two heroes, leading up to the expedition against Ḫu(m)baba. Lines 38–40 of [84]column 2 of the Assyrian version correspond to lines 174–177 of the Pennsylvania tablet, and lines 44–50 to lines 192–221. It would seem that Gish proceeds stealthily at night to go to the goddess Ishḫara, who lies on a couch in the bît êmuti , the “family house” Assyrian version, Tablet IV, 2. 46–48). He encounters Enkidu in the street, and the latter blocks Gish’s path, puts his foot in the gate leading to the house where the goddess is, and thus prevents Gish from entering. Thereupon the two have a fierce encounter in which Gish is worsted. The meaning of the episode itself is not clear. Does Enkidu propose to deprive Gish, here viewed as a god (cf. line 190 of the Pennsylvania tablet = Assyrian version, Tablet I, 4, 45, “like a god”), of his spouse, the goddess Ishḫara—another form of Ishtar? Or are the two heroes, the one a counterpart of the other, contesting for the possession of a goddess? Is it in this scene that Enkidu becomes the “rival” (me-iḫ-rù, line 191 of the Pennsylvania tablet) of the divine Gish? We must content ourself with having obtained through the Pennsylvania tablet a clearer indication of the occasion of the fight between the two heroes, and leave the further explanation of the episode till a fortunate chance may throw additional light upon it. There is perhaps a reference to the episode in the Assyrian version, Tablet II, 3b, 35–36. Line 196. For i-na-ag-šá-am (from nagâšu), Langdon proposes the purely fanciful “embracing her in sleep,” whereas it clearly means “he approaches.” Cf. Muss-Arnolt, Assyrian Dictionary, page 645a. Lines 197–200 appear to correspond to Tablet IV, 2, 35–37, of the Assyrian version, though not forming a complete parallel. We may therefore supply at the beginning of line 35 of the Assyrian version [ittaziz] Enkidu, corresponding to line 197 of the Pennsylvania tablet. Line 36 of IV, 2, certainly appears to correspond to line 200 (dan-nu-ti = da-na-ni-iš-šú). Line 208. The first sign looks more like šar, though ur is possible. Line 211 is clearly a description of Enkidu, as is shown by a comparison with the Assyrian version I, 2, 37: [pi]-ti-ik pi-ir-ti-šú uḫ-tan-na-ba kima dNidaba, “The form of his hair sprouted like wheat.” We must therefore supply Enkidu in the preceding line. Tablet IV, 4, 6, of the Assyrian version also contains a reference to the flowing hair of Enkidu. [85] Line 212. For the completion of the line cf. Harper, Assyrian and Babylonian Letters, No. 214. Line 214. For ribîtu mâti see the note above to line 28 of column 1. Lines 215–217 correspond almost entirely to the Assyrian version IV, 2, 46–48. The variations ki-ib-su in place of šêpu, and kima lîm, “like oxen,” instead of ina bâb êmuti (repeated from line 46), ana šurûbi for êribam, are slight though interesting. The Assyrian version shows that the “gate” in line 215 is “the gate of the family house” in which the goddess Ishḫara lies. Lines 218–228. The detailed description of the fight between the two heroes is only partially preserved in the Assyrian version. Line 218. li-i-im is evidently to be taken as plural here as in line 224, just as su-ḳi-im (lines 27 and 175), ri-bi-tim (lines 4, 28, etc.), tarbaṣim (line 74), aṣṣamim (line 98) are plural forms. Our text furnishes, as does also the Yale tablet, an interesting illustration of the vacillation in the Hammurabi period in the twofold use of im: (a) as an indication of the plural (as in Hebrew), and (b) as a mere emphatic ending (lines 63, 73, and 232), which becomes predominant in the post-Hammurabi age. Line 227. Gilgamesh is often represented on seal cylinders as kneeling, e.g., Ward Seal Cylinders Nos. 159, 160, 165. Cf. also Assyrian version V, 3, 6, where Gilgamesh is described as kneeling, though here in prayer. See further the commentary to the Yale tablet, line 215. Line 229. We must of course read uz-za-šú, “his anger,” and not uṣ-ṣa-šú, “his javelin,” as Langdon does, which gives no sense. Line 231. Langdon’s note is erroneous. He again misses the point. The stem of the verb here as in line 230 (i-ni-iḫ) is the common nâḫu, used so constantly in connection with pašâḫu, to designate the cessation of anger. Line 234. ištên applied to Gish designates him of course as “unique,” not as “an ordinary man,” as Langdon supposes. Line 236. On this title “wild cow of the stall” for Ninsun, see Poebel in OLZ 1914, page 6, to whom we owe the correct view regarding the name of Gilgamesh’s mother. Line 238. mu-ti here cannot mean “husband,” but “man” in [86]general. See above note to line 107. Langdon’s strange misreading ri-eš-su for ri-eš-ka (“thy head”) leads him again to miss the point, namely that Enkidu comforts his rival by telling him that he is destined for a career above that of the ordinary man. He is to be more than a mere prize fighter; he is to be a king, and no doubt in the ancient sense, as the representative of the deity. This is indicated by the statement that the kingship is decreed for him by Enlil. Similarly, Ḫu(m)baba or Ḫuwawa is designated by Enlil to inspire terror among men (Assyrian version, Tablet IV, 5, 2 and 5), i-šim-šú dEnlil = Yale tablet, l. 137, where this is to be supplied. This position accorded to Enlil is an important index for the origin of the Epic, which is thus shown to date from a period when the patron deity of Nippur was acknowledged as the general head of the pantheon. This justifies us in going back several centuries at least before Hammurabi for the beginning of the Gilgamesh story. If it had originated in the Hammurabi period, we should have had Marduk introduced instead of Enlil. Line 242. As has been pointed out in the corrections to the text (Appendix), šú-tu-ur can only be III, 1, from atâru, “to be in excess of.” It is a pity that the balance of the line is broken off, since this is the first instance of a colophon beginning with the term in question. In some way šutûr must indicate that the copy of the text has been “enlarged.” It is tempting to fill out the line šú-tu-ur e-li [duppi labiri], and to render “enlarged from an original,” as an indication of an independent recension of the Epic in the Hammurabi period. All this, however, is purely conjectural, and we must patiently hope for more tablets of the Old Babylonian version to turn up. The chances are that some portions of the same edition as the Yale and Pennsylvania tablets are in the hands of dealers at present or have been sold to European museums. The war has seriously interfered with the possibility of tracing the whereabouts of groups of tablets that ought never to have been separated. [87] Yale Tablet. Transliteration. (About ten lines missing.) Col. I. 11.................. [ib]-ri(?) 12[mi-im-ma(?) šá(?)]-kú-tu wa(?)-ak-rum 13[am-mi-nim] ta-aḫ-ši-iḫ 14[an-ni]-a-am [e-pi]-šá-am 15...... mi-im[-ma šá-kú-tu(?)]ma- 16di-iš 17[am-mi]-nim [taḫ]-ši-iḫ 18[ur(?)]-ta-du-ú [a-na ki-i]š-tim 19ši-ip-ra-am it-[ta-šú]-ú i-na [nišê] 20it-ta-áš-šú-ú-ma 21i-pu-šú ru-ḫu-tam 22.................. uš-ta-di-nu 23............................. bu 24............................... (About 17 lines missing.) 40.............. nam-........ 41.................... u ib-[ri] ..... 42.............. ú-na-i-du ...... 43[zi-ik]-ra-am ú-[tí-ir]-ru 44[a-na] ḫa-ri-[im]-tim 45[i]-pu(?)-šú a-na sa-[ka]-pu-ti Col. II. (About eleven lines missing.) 57... šú(?)-mu(?) ............... 58ma-ḫi-ra-am [šá i-ši-šú] 59šú-uk-ni-šum-[ma] ............... 60la-al-la-ru-[tu] .................. 61um-mi d-[Giš mu-di-a-at ka-la-ma] 62i-na ma-[ḫar dŠamaš i-di-šá iš-ši][88] 63šá ú 64i-na- an(?)-[na am-mi-nim] 65ta-[aš-kun(?) a-na ma-ri-ia li-ib-bi la] 66ṣa-[li-la te-mid-su] 67............................. (About four lines missing.) 72i-na [šá dEn-ki-dũ im-la-a] di-[im-tam] 73il-[pu-ut li]-ib-ba-šú-[ma] 74[zar-biš(?)] uš-ta-ni-[iḫ] 75[i-na šá dEn]-ki-dũ im-la-a di-im-tam 76[il-pu-ut] li-ib-ba-šú-ma 77[zar-biš(?)] uš-ta-ni-[iḫ] 78[dGiš ú-ta]-ab-bil pa-ni-šú 79[iz-za-kar-am] a-na dEn-ki-dũ 80[ib-ri am-mi-nim] i-na-ka 81[im-la-a di-im]-tam 82[il-pu-ut li-ib-bi]-ka 83[zar-biš tu-uš-ta]-ni-iḫ 84[dEn-ki-dũ pi-šú i-pu-šá]-am-ma 85iz-za-[kàr-am] a-na dGiš 86ta-ab-bi-a-tum ib-ri 87uš-ta-li-pa da-1da-ni-ia 88a-ḫa-a-a ir-ma-a-ma 89e-mu-ki i-ni-iš 90dGiš pi-šú i-pu-šá-am-ma 91iz-za-kàr-am a-na dEn-ki-dũ (About four lines missing.) Col. III. 96..... [a-di dḪu]-wa-wa da-pi-nu 97.................. ra-[am(?)-ma] 98................ [ú-ḫal]- li-ik 99[lu-ur-ra-du a-na ki-iš-ti šá] iserini[89] 100............ lam(?) ḫal-bu 101............ [li]-li-is-su 102.............. lu(?)-up-ti-šú 103dEn-ki-dũ pi-šú i-pu-šá-am-ma 104iz-za-kàr-am a-na dGiš 105i-di-ma ib-ri i-na šadî(-i) 106i-nu-ma at-ta-la-ku it-ti bu-lim 107a-na ištên(-en) kas-gíd-ta-a-an nu-ma-at ki-iš-tum 108[e-di-iš(?)] ur-ra-du a-na libbi-šá 109d[Ḫu-wa]-wa ri-ig-ma-šú a-bu-bu 110pi-[šú] dBil-gi-ma 111na-pi-iš-šú mu-tum 112am-mi-nim ta-aḫ-ši-iḫ 113an-ni-a-am e-pi-šá-am 114ga-[ba]-al-la ma-ḫa-ar 115[šú]-pa-at dḪu-wa-wa 116(d)Giš pi-šú i-pu-šá-am-ma 117[iz-za-k]àr-am a-na dEn-ki-dũ 118....... su(?)-lu-li a-šá-ki2-šá 119............. [i-na ki-iš]-tim 120............................... 121ik(?) ......................... 122a-na .......................... 123mu-šá-ab [dḪu-wa-wa] ....... 124ḫa-aṣ-si-nu ................. 125at-ta lu(?) ................. 126a-na-ku lu-[ur-ra-du a-na ki-iš-tim] 127dEn-ki-dũ pi-šú i-pu-[šá-am-ma] 128iz-za-kàr-am a-na [dGiš] 129ki-i ni[il]-la-ak [iš-te-niš(?)] 130a-na ki-iš-ti [šá iṣerini] 131na-ṣi-ir-šá dGiš muḳ-[tab-lu] 132da-a-an la ṣa[-li-lu(?)] 133dḪu-wa-wa dpi-ir-[ḫu ša (?)][90] 134dAdad iš .......... 135šú-ú .................. Col. IV. 136áš-šúm šú-ul-lu-m[u ki-iš-ti šáiṣerini] 137pu-ul-ḫi-a-tim 7 [šú(?) i-šim-šú dEnlil] 138dGiš pi-šú i-pu [šá-am-ma] 139iz-za-kàr-am a-na [dEn-ki-dũ] 140ma-an-nu ib-ri e-lu-ú šá-[ru-ba(?)] 141i-ṭib-ma it-ti dŠamaš da-ri-iš ú-[me-šú] 142a-we-lu-tum ba-ba-nu ú-tam-mu-šá-[ma] 143mi-im-ma šá i-te-ni-pu-šú šá-ru-ba 144at-ta an-na-nu-um-ma ta-dar mu-tam 145ul iš-šú da-na-nu ḳar-ra-du-ti-ka 146lu-ul-li-ik-ma i-na pa-ni-ka 147pi-ka li-iš-si-a-am ṭi-ḫi-e ta-du-ur 148šum-ma am-ta-ḳu-ut šú-mi lu-uš-zi-iz 149dGiš mi3-it-ti dḪu-wa-wa da-pi-nim 150il(?)-ḳu-ut iš-tu 151i-wa-al-dam-ma tar-bi-a i-na šam-mu(?) Il(?) 152iš-ḫi-it-ka-ma la-bu ka-la-ma ti-di 153it- ku(?) ..... [il(?)]-pu-tu-(?) ma ..... 154.............. ka-ma 155.............. ši pi-ti 156............ ki-ma re’i(?) na-gi-la sa-rak-ti 157.... [ta-šá-s]i-a-am tu-lim-mi-in li-ib-bi 158[ga-ti lu]-uš-ku-un-ma 159[lu-u-ri]-ba-am iṣerini[91] 160[šú-ma sá]-ṭa-ru-ú a-na-ku lu-uš-ta-ak-na 161[pu-tu-ku(?)] ib-ri a-na ki-iš-ka-tim lu-mu-ḫa 162[be-le-e li-iš-]-pu-ku i-na maḫ-ri-ni 163[pu-tu]-ku a-na ki-iš-ka-ti-i i-mu-ḫu 164wa-áš-bu uš-ta-da-nu um-mi-a-nu 165pa-ši iš-pu-ku ra-bu-tim 166ḫa-aṣ-si-ni 3 biltu-ta-a-an iš-tap-ku 167pa-aṭ-ri iš-pu-ku ra-bu-tim 168me-še-li-tum 2 biltu-ta-a-an 169ṣi-ip-ru 30 ma-na-ta-a-an šá a-ḫi-ši-na 170išid(?) pa-aṭ-ri 30 ma-na-ta-a-an ḫuraṣi 171[d]Giš ù [dEn-ki-]dũ 10 biltu-ta-a-an šá-ak-nu] 172.... ul-la . .[Uruk]ki 7 i-di-il-šú 173...... iš-me-ma um-ma-nu ib-bi-ra 174[uš-te-(?)]-mi-a i-na sûḳi šá Urukki ri-bi-tim 175...... [u-še(?)]-ṣa-šú dGis 176[ina sûḳi šá(?) Urukki] ri-bi-tim 177[dEn-ki-dũ(?) ú]-šá-ab i-na maḫ-ri-šú 178..... [ki-a-am(?) i-ga]-ab-bi 179[........ Urukki ri]-bi-tim 180 [ma-ḫa-ar-šú] Col. V. 181dGiš šá i-ga-ab-bu-ú lu-mu-ur 182šá šú-um-šú it-ta-nam-ma-la ma-ta-tum 183lu-uk-šú-su-ma i-na ki-iš-ti iṣerini 184ki-ma da-an-nu pi-ir-ḫu-um šá Urukki[92] 185lu-ši-eš-mi ma-tam 186ga-ti lu-uš-ku-un-ma lu-uk-[šú]4-su-ma iṣerini 187šú-ma šá-ṭa-ru-ú a-na-ku lu-uš-tak-nam 188ši-bu-tum šá Urukki ri-bi-tim 189zi-ik-ra ú-ti-ir-ru a-na dGiš 190ṣi-iḫ-ri-ti-ma dGiš libbi-ka na-ši-ka 191mi-im-ma šá te-te-ni-pu-šú la ti-di 192ni-ši-im-me-ma dḪu-wa-wa šá-nu-ú bu-nu-šú 193ma-an-nu-um [uš-tam]-ḫa-ru ka-ak-ki-šú 194a-na ištên(-en) [kas-gíd-ta-a]-an nu-ma-at kišti 195ma-an-nu šá [ur-ra]-du a-na libbi-šá 196dḪu-wa-wa ri-ig-ma-šú a-bu-bu 197pi-šú dBil-gi-ma na-pi-su mu-tum 198am-mi-nim taḫ-ši-iḫ an-ni-a-am e-pi-šá 199ga-ba-al-la ma-ḫa-ar šú-pa-at dḪu-wa-wa 200iš-me-e-ma dGiš zi-ki-ir ma-li-[ki]-šú 201ip-pa-al-sa-am-ma i-ṣi-iḫ a-na ib-[ri-šú] 202i-na-an-na ib-[ri] ki-a-am [a-ga-ab-bi] 203a-pa-al-aḫ-šú-ma a-[al-la-ak a-na kišti] 204[lu]ul-[lik it-ti-ka a-na ki-iš-ti iṣerini(?)] (About five lines missing.) 210........................ -ma 211li ............... -ka[93] 212ilu-ka li(?) ..............-ka 213ḫarrana li-šá-[tir-ka a-na šú-ul-mi] 214a-na kar šá [Urukki ri-bi-tim] 215ka-mi-is-ma dGiš [ma-ḫa-ar dŠamaš(?)] 216a-wa-at i-ga-ab- [bu-šú-ma] 217a-al-la-ak dŠamaš katâ-[ka a-ṣa-bat] 218ul-la-nu lu-uš-li-ma na-pi-[iš-ti] 219te-ir-ra-an-ni a-na kar i-[na Urukki] 220ṣi-il-[la]m šú-ku-un [a-na ia-a-ši(?)] 221iš-si-ma dGiš ib-[ri.....] 222te-ir-ta-šú .......... 223is(?) .............. 224tam ................ 225........................ 226i-nu(?)-[ma] .................. (About two lines missing.) Col. VI. 229[a-na-ku] dGiš [i-ik]-ka-di ma-tum 230........... ḫarrana šá la al-[kam] ma-ti-ma 231.... a-ka-lu ..... la(?) i-di 232[ul-la-nu] lu-uš-li-[mu] a-na-ku 233[lu-ud-lul]-ka i-na [ḫ]u-ud li-ib-bi 234...... [šú]-ḳu-ut-[ti] la-li-ka 235[lu-še-šib(?)] - ka i-na kussêmeš 236....................... ú-nu-su 237[bêlêmeš(?)ú-ti-ir]-ru ra-bu-tum 238[ka-aš-tum] ù iš-pa-tum 239[i-na] ga-ti iš-ku-nu 240[il-]te-ki pa-ši 241....... -ri iš-pa-as-su[94] 242..... [a-na] ili šá-ni-tam 243[it-ti pa(?)] - tar-[šú] i-na ši-ip-pi-šú 244........ i-ip-pu-šú a-la-kam 245[ša]-niš ú-ga-ra-bu dGiš 246[a-di ma]-ti tu-ut-te-ir a-na libbi Urukki 247[ši-bu]-tum i-ka-ra-bu-šú 248[a-na] ḫarrani i-ma-li-ku dGiš 249[la t]a-at-kal dGiš a-na e-[mu]-ḳi-ka 250[a-]ka-lu šú-wa-ra-ma ú-ṣur ra-ma-an-ka 251[li]-il-lik dEn-ki-dũ i-na pa-ni-ka 252[ur-ḫa]-am a-we-ir a-lik ḫarrana(-na) 253[a-di] šá kišti ni-ri-bi-tim 254[šá(?)] [d]Ḫu-wa-wa ka-li-šú-nu ši-ip-pi-iḫ(?)-šú 255[ša(?)a-lik] maḫ-ra tap-pa-a ú-šá-lim 256[ḫarrana](-na)-šú šú-wa-ra-[ma ú-ṣur ra-ma-na-ka] 257[li-šak-šid]-ka ir-[ni-ta]-ka dŠamaš 258[ta]-ak-bi-a-at pi-ka li-kal-li-ma i-na-ka 259li-ip-ti-ḳu pa-da-nam pi-ḫi-tam 260ḫarrana li-iš-ta-zi-ik a-na ki-ib-si-ka 261šá-di-a li-iš-ta-zi-ik a-na šêpi-ka 262mu-ši-it-ka aw-a-at ta-ḫa-du-ú 263li-ib-la-ma dLugal-ban-da li-iz-zi-iz-ka[95] 264i-na ir-ni-ti-ka 265ki-ma ṣi-iḫ-ri ir-ni-ta-ka-ma luš-mida(-da) 266i-na na-ri šá dḪu-wa-wa šá tu-ṣa-ma-ru 267mi-zi ši-pi-ka 268i-na bat-ba-ti-ka ḫi-ri bu-ur-tam 269lu-ka-a-a-nu mê ellu i-na na-di-ka 270[ka-]su-tim me-e a-na dŠamaš ta-na-di 271[li-iš]ta-ḫa-sa-as dLugal-ban-da 272[dEn-ki-]dũ pi-su i-pu-šá-am-ma, iz-za-kàr a-na dGiš 273[is(?)]-tu(?) ta-áš-dan-nu e-pu-uš a-la-kam 274[la pa]la-aḫ libbi-ka ia-ti tu-uk-la-ni 275[šú-ku-]un i-di-a-am šú-pa-as-su 276[ḫarrana(?)]šá dḪu-wa-wa it-ta-la-ku 277.......... ki-bi-ma te-[ir]-šú-nu-ti (Three lines missing.) L.E. 281.............. nam-ma-la 282............... il-li-ku it-ti-ia 283............... ba-ku-nu-ši-im 284......... [ul]-la(?)-nu i-na ḫu-ud li-ib-bi 285[i-na še-me-e] an-ni-a ga-ba-šú 286e-diš ḫarrana(?) uš-te-[zi-ik] 287a-lik dGiš lu-[ul-lik a-na pa-ni-ka] 288li-lik il-ka .......... 289li-šá-ak-lim-[ka ḫarrana] ...... 290dGiš ù[dEn-ki-dũ] ....... 291mu-di-eš .......... 292bi-ri-[su-nu] ........ [87] Translation. (About ten lines missing.) Col. I. 11.................. (my friend?) 12[Something] that is exceedingly difficult, 13[Why] dost thou desire 14[to do this?] 15.... something (?) that is very [difficult (?)], 16[Why dost thou] desire 17[to go down to the forest]? 18A message [they carried] among [men] 19They carried about. 20They made a .... 21.............. they brought 22.............................. 23.............................. (About 17 lines missing.) 40............................. 41................... my friend 42................ they raised ..... 43answer [they returned.] 44[To] the woman 45They proceeded to the overthrowing Col. II. (About eleven lines missing.) 57.......... name(?) ............. 58[The one who is] a rival [to him] 59subdue and ................ 60Wailing ................ 61The mother [of Gišh, who knows everything] 62Before [Shamash raised her hand][88] 63Who 64Now(?) [why] 65hast thou stirred up the heart for my son, 66[Restlessness imposed upon him (?)] 67............................ (About four lines missing.) 72The eyes [of Enkidu filled with tears]. 73[He clutched] his heart; 74[Sadly(?)] he sighed. 75[The eyes of En]kidu filled with tears. 76[He clutched] his heart; 77[Sadly(?)] he sighed. 78The face [of Gišh was grieved]. 79[He spoke] to Enkidu: 80[“My friend, why are] thy eyes 81[Filled with tears]? 82Thy [heart clutched] 83Dost thou sigh [sadly(?)]?” 84[Enkidu opened his mouth] and 85spoke to Gišh: 86“Attacks, my friend, 87have exhausted my strength(?). 88My arms are lame, 89my strength has become weak.” 90Gišh opened his mouth and 91spoke to Enkidu: (About four lines missing.) Col. III. 96..... [until] Ḫuwawa, [the terrible], 97........................ 98............ [I destroyed]. 99[I will go down to the] cedar forest,[89] 100................... the jungle 101............... tambourine (?) 102................ I will open it. 103Enkidu opened his mouth and 104spoke to Gišh: 105“Know, my friend, in the mountain, 106when I moved about with the cattle 107to a distance of one double hour into the heart of the forest, 108[Alone?] I penetrated within it, 109[To] Ḫuwawa, whose roar is a flood, 110whose mouth is fire, 111whose breath is death. 112Why dost thou desire 113To do this? 114To advance towards 115the dwelling(?) of Ḫuwawa?” 116Gišh opened his mouth and 117[spoke to Enkidu: 118”... [the covering(?)] I will destroy. 119....[in the forest] 120.................... 121.................... 122To ................. 123The dwelling [of Ḫuwawa] 124The axe .......... 125Thou .......... 126I will [go down to the forest].” 127Enkidu opened his mouth and 128spoke to [Gish:] 129“When [together(?)] we go down 130To the [cedar] forest, 131whose guardian, O warrior Gish, 132a power(?) without [rest(?)], 133Ḫuwawa, an offspring(?) of ....[90] 134Adad ...................... 135He ........................ Col. IV. 136To keep safe [the cedar forest], 137[Enlil has decreed for it] seven-fold terror.” 138Gish [opened] his mouth and 139spoke to [Enkidu]: 140“Whoever, my friend, overcomes (?) [terror(?)], 141it is well (for him) with Shamash for the length of [his days]. 142Mankind will speak of it at the gates. 143Wherever terror is to be faced, 144Thou, forsooth, art in fear of death. 145Thy prowess lacks strength. 146I will go before thee. 147Though thy mouth calls to me; “thou art afraid to approach.” 148If I fall, I will establish my name. 149Gish, the corpse(?) of Ḫuwawa, the terrible one, 150has snatched (?) from the time that 151My offspring was born in ...... 152The lion restrained (?) thee, all of which thou knowest. 153........................ 154.............. thee and 155................ open (?) 156........ like a shepherd(?) ..... 157[When thou callest to me], thou afflictest my heart. 158I am determined 159[to enter] the cedar forest.[91] 160I will, indeed, establish my name. 161[The work(?)], my friend, to the artisans I will entrust. 162[Weapons(?)] let them mould before us.” 163[The work(?)] to the artisans they entrusted. 164A dwelling(?) they assigned to the workmen. 165Hatchets the masters moulded: 166Axes of 3 talents each they moulded. 167Lances the masters moulded; 168Blades(?) of 2 talents each, 169A spear of 30 mina each attached to them. 170The hilt of the lances of 30 mina in gold 171Gish and [Enki]du were equipped with 10 talents each 172.......... in Erech seven its .... 173....... the people heard and .... 174[proclaimed(?)] in the street of Erech of the plazas. 175..... Gis [brought him out(?)] 176[In the street (?)] of Erech of the plazas 177[Enkidu(?)] sat before him 178..... [thus] he spoke: 179”........ [of Erech] of the plazas 180............ [before him] Col. V. 181Gish of whom they speak, let me see! 182whose name fills the lands. 183I will lure him to the cedar forest, 184Like a strong offspring of Erech.[92] 185I will let the land hear (that) 186I am determined to lure (him) in the cedar (forest)5. 187A name I will establish.” 188The elders of Erech of the plazas 189brought word to Gish: 190“Thou art young, O Gish, and thy heart carries thee away. 191Thou dost not know what thou proposest to do. 192We hear that Huwawa is enraged. 193Who has ever opposed his weapon? 194To one [double hour] in the heart of the forest, 195Who has ever penetrated into it? 196Ḫuwawa, whose roar is a deluge, 197whose mouth is fire, whose breath is death. 198Why dost thou desire to do this? 199To advance towards the dwelling (?) of Ḫuwawa?” 200Gish heard the report of his counsellors. 201He saw and cried out to [his] friend: 202“Now, my friend, thus [I speak]. 203I fear him, but [I will go to the cedar forest(?)]; 204I will go [with thee to the cedar forest]. (About five lines missing.) 210.............................. 211May ................... thee[93] 212Thy god may (?) ........ thee; 213On the road may he guide [thee in safety(?)]. 214At the rampart of [Erech of the plazas], 215Gish kneeled down [before Shamash(?)], 216A word then he spoke [to him]: 217“I will go, O Shamash, [thy] hands [I seize hold of]. 218When I shall have saved [my life], 219Bring me back to the rampart [in Erech]. 220Grant protection [to me ?]!” 221Gish cried, ”[my friend] ...... 222His oracle .................. 223........................ 224........................ 225........................ 226When (?) (About two lines missing.) Col. VI. 229”[I(?)] Gish, the strong one (?) of the land. 230...... A road which I have never [trodden]; 231........ food ...... do not (?) know. 232[When] I shall have succeeded, 233[I will praise] thee in the joy of my heart, 234[I will extol (?)] the superiority of thy power, 235[I will seat thee] on thrones.” 236.................. his vessel(?) 237The masters [brought the weapons (?)]; 238[bow] and quiver 239They placed in hand. 240[He took] the hatchet. 241................. his quiver.[94] 242..... [to] the god(?) a second time 243[With his lance(?)] in his girdle, 244......... they took the road. 245[Again] they approached Gish! 246”[How long] till thou returnest to Erech?” 247[Again the elders] approached him. 248[For] the road they counselled Gis: 249“Do [not] rely, O Gish, on thy strength! 250Provide food and save thyself! 251Let Enkidu go before thee. 252He is acquainted with the way, he has trodden the road 253[to] the entrance of the forest. 254of Ḫuwawa all of them his ...... 255[He who goes] in advance will save the companion. 256Provide for his [road] and [save thyself]! 257(May) Shamash [carry out] thy endeavor! 258May he make thy eyes see the prophecy of thy mouth. 259May he track out (for thee) the closed path! 260May he level the road for thy treading! 261May he level the mountain for thy foot! 262During thy night6 the word that wilt rejoice 263may Lugal-banda convey, and stand by thee[95] 264in thy endeavor! 265Like a youth may he establish thy endeavor! 266In the river of Ḫuwawa as thou plannest, 267wash thy feet! 268Round about thee dig a well! 269May there be pure water constantly for thy libation 270Goblets of water pour out to Shamash! 271[May] Lugal-banda take note of it!” 272[Enkidu] opened his mouth and spoke to Gish: 273”[Since thou art resolved] to take the road. 274Thy heart [be not afraid,] trust to me! 275[Confide] to my hand his dwelling(?)!” 276[on the road to] Ḫuwawa they proceeded. 277....... command their return (Three lines missing.) L.E. 281............... were filled. 282.......... they will go with me. 283............................... 284.................. joyfully. 285[Upon hearing] this word of his, 286Alone, the road(?) [he levelled]. 287“Go, O Gish [I will go before thee(?)]. 288May thy god(?) go ......... 289May he show [thee the road !] ..... 290Gish and [Enkidu] 291Knowingly .................... 292Between [them] ................ [96]Lines 13–14 (also line 16). See for the restoration, lines 112–13. Line 62. For the restoration, see Jensen, p. 146 (Tablet III, 2a,9.) Lines 64–66. Restored on the basis of the Assyrian version, ib. line 10. Line 72. Cf. Assyrian version, Tablet IV, 4, 10, and restore at the end of this line di-im-tam as in our text, instead of Jensen’s conjecture. Lines 74, 77 and 83. The restoration zar-biš, suggested by the Assyrian version, Tablet IV, 4, 4. Lines 76 and 82. Cf. Assyrian version, Tablet VIII, 3, 18. Line 78. (ú-ta-ab-bil from abâlu, “grieve” or “darkened.” Cf. uš-ta-kal (Assyrian version, ib. line 9), where, perhaps, we are to restore it-ta-[bil pa-ni-šú]. Line 87. uš-ta-li-pa from elêpu, “exhaust.” See Muss-Arnolt, Assyrian Dictionary, p. 49a. Line 89. Cf. Assyrian version, ib. line 11, and restore the end of the line there to i-ni-iš, as in our text. Line 96. For dapinu as an epithet of Ḫuwawa, see Assyrian version, Tablet III, 2a, 17, and 3a, 12. Dapinu occurs also as a description of an ox (Rm 618, Bezold, Catalogue of the Kouyunjik Tablets, etc., p. 1627). Line 98. The restoration on the basis of ib. III, 2a, 18. Lines 96–98 may possibly form a parallel to ib. lines 17–18, which would then read about as follows: “Until I overcome Ḫuwawa, the terrible, and all the evil in the land I shall have destroyed.” At the same time, it is possible that we are to restore [lu-ul]-li-ik at the end of line 98. Line 101. lilissu occurs in the Assyrian version, Tablet IV, 6, 36. Line 100. For ḫalbu, “jungle,” see Assyrian version, Tablet V, 3, 39 (p. 160). Lines 109–111. These lines enable us properly to restore Assyrian version, Tablet IV, 5, 3 = Haupt’s edition, p. 83 (col. 5, 3). No doubt the text read as ours mu-tum (or mu-u-tum) na-pis-su. Line 115. šupatu, which occurs again in line 199 and also line 275.šú-pa-as-su (= šupat-su) must have some such meaning as [97]“dwelling,” demanded by the context. [Dhorme refers me to OLZ 1916, p. 145]. Line 129. Restored on the basis of the Assyrian version, Tablet IV, 6, 38. Line 131. The restoration muḳtablu, tentatively suggested on the basis of CT XVIII, 30, 7b, where muḳtablu, “warrior,” appears as one of the designations of Gilgamesh, followed by a-lik pa-na, “the one who goes in advance,” or “leader”—the phrase so constantly used in the Ḫuwawa episode. Line 132. Cf. Assyrian version, Tablet I, 5, 18–19. Lines 136–137. These two lines restored on the basis of Jensen IV, 5, 2 and 5. The variant in the Assyrian version, šá niše (written Ukumeš in one case and Lumeš in the other), for the numeral 7 in our text to designate a terror of the largest and most widespread character, is interesting. The number 7 is similarly used as a designation of Gilgamesh, who is called Esigga imin, “seven-fold strong,” i.e., supremely strong (CT XVIII, 30, 6–8). Similarly, Enkidu, ib. line 10, is designated a-rá imina, “seven-fold.” Line 149. A difficult line because of the uncertainty of the reading at the beginning of the following line. The most obvious meaning of mi-it-tu is “corpse,” though in the Assyrian version šalamtu is used (Assyrian version, Tablet V, 2, 42). On the other hand, it is possible—as Dr. Lutz suggested to me—that mittu, despite the manner of writing, is identical with miṭṭú, the name of a divine weapon, well-known from the Assyrian creation myth (Tablet IV, 130), and other passages. The combination miṭ-ṭu šá-ḳu-ú-, “lofty weapon,” in the Bilingual text IV, R², 18 No. 3, 31–32, would favor the meaning “weapon” in our passage, since [šá]-ḳu-tu is a possible restoration at the beginning of line 150. However, the writing mi-it-ti points too distinctly to a derivative of the stem mâtu, and until a satisfactory explanation of lines 150–152 is forthcoming, we must stick to the meaning “corpse” and read the verb il-ḳu-ut. Line 152. The context suggests “lion” for the puzzling la-bu. Line 156. Another puzzling line. Dr. Clay’s copy is an accurate reproduction of what is distinguishable. At the close of the line there appears to be a sign written over an erasure. Line 158. [ga-ti lu-]uš-kun as in line 186, literally, “I will place my hand,” i.e., I purpose, I am determined. [98] Line 160. The restoration on the basis of the parallel line 187. Note the interesting phrase, “writing a name” in the sense of acquiring “fame.” Line 161. The kiškattê, “artisans,” are introduced also in the Assyrian version, Tablet VI, 187, to look at the enormous size and weight of the horns of the slain divine bull. See for other passages Muss-Arnolt Assyrian Dictionary, p. 450b. At the beginning of this line, we must seek for the same word as in line 163. Line 162. While the restoration belê, “weapon,” is purely conjectural, the context clearly demands some such word. I choose belê in preference to kakkê, in view of the Assyrian version, Tablet VI, 1. Line 163. Putuku (or putukku) from patâku would be an appropriate word for the fabrication of weapons. Line 165. The rabûtim here, as in line 167, I take as the “master mechanics” as contrasted with the ummianu, “common workmen,” or journeymen. A parallel to this forging of the weapons for the two heroes is to be found in the Sumerian fragment of the Gilgamesh Epic published by Langdon, Historical and Religious Texts from the Temple Library of Nippur (Munich, 1914), No. 55, 1–15. Lines 168–170 describe the forging of the various parts of the lances for the two heroes. The ṣipru is the spear point Muss-Arnolt, Assyrian Dictionary, p. 886b; the išid paṭri is clearly the “hilt,” and the mešelitum I therefore take as the “blade” proper. The word occurs here for the first time, so far as I can see. For 30 minas, see Assyrian version, Tablet VI, 189, as the weight of the two horns of the divine bull. Each axe weighing 3 biltu, and the lance with point and hilt 3 biltu we would have to assume 4 biltu for each pašu, so as to get a total of 10 biltu as the weight of the weapons for each hero. The lance is depicted on seal cylinders representing Gilgamesh and Enkidu, for example, Ward, Seal Cylinders, No. 199, and also in Nos. 184 and 191 in the field, with the broad hilt; and in an enlarged form in No. 648. Note the clear indication of the hilt. The two figures are Gilgamesh and Enkidu—not two Gilgameshes, as Ward assumed. See above, page 34. A different weapon is the club or mace, as seen in Ward, Nos. 170 and 173. This appears also to be the weapon which Gilgamesh holds in his hand on the colossal figure from the palace of Sargon (Jastrow, Civilization of [99]Babylonia and Assyria, Pl. LVII), though it has been given a somewhat grotesque character by a perhaps intentional approach to the scimitar, associated with Marduk (see Ward, Seal Cylinders, Chap. XXVII). The exact determination of the various weapons depicted on seal-cylinders merits a special study. Line 181. Begins a speech of Ḫuwawa, extending to line 187, reported to Gish by the elders (line 188–189), who add a further warning to the youthful and impetuous hero. Line 183. lu-uk-šú-su (also l. 186), from akâšu, “drive on” or “lure on,” occurs on the Pennsylvania tablet, line 135, uk-ki-ši, “lure on” or “entrap,” which Langdon erroneously renders “take away” and thereby misses the point completely. See the comment to the line of the Pennsylvania tablet in question. Line 192. On the phrase šanû bunu, “change of countenance,” in the sense of “enraged,” see the note to the Pennsylvania tablet, l.31. Line 194. nu-ma-at occurs in a tablet published by Meissner, Altbabyl. Privatrecht, No. 100, with bît abi, which shows that the total confine of a property is meant; here, therefore, the “interior” of the forest or heart. It is hardly a “by-form” of nuptum as Muss-Arnolt, Assyrian Dictionary, p. 690b, and others have supposed, though nu-um-tum in one passage quoted by Muss-Arnolt, ib. p. 705a, may have arisen from an aspirate pronunciation of the p in nubtum. Line 215. The kneeling attitude of prayer is an interesting touch. It symbolizes submission, as is shown by the description of Gilgamesh’s defeat in the encounter with Enkidu (Pennsylvania tablet, l. 227), where Gilgamesh is represented as forced to “kneel” to the ground. Again in the Assyrian version, Tablet V, 4, 6, Gilgamesh kneels down (though the reading ka-mis is not certain) and has a vision. Line 229. It is much to be regretted that this line is so badly preserved, for it would have enabled us definitely to restore the opening line of the Assyrian version of the Gilgamesh Epic. The fragment published by Jeremias in his appendix to his Izdubar-Nimrod, Plate IV, gives us the end of the colophon line to the Epic, reading ……… di ma-a-ti (cf. ib., Pl. I, 1. … a-ti). Our text evidently reproduces the same phrase and enables us to supply ka, as well as [100]the name of the hero Gišh of which there are distinct traces. The missing word, therefore, describes the hero as the ruler, or controller of the land. But what are the two signs before ka? A participial form from pakâdu, which one naturally thinks of, is impossible because of the ka, and for the same reason one cannot supply the word for shepherd (nakidu). One might think of ka-ak-ka-du, except that kakkadu is not used for “head” in the sense of “chief” of the land. I venture to restore [i-ik-]ka-di, “strong one.” Our text at all events disposes of Haupt’s conjecture iš-di ma-a-ti (JAOS 22, p. 11), “Bottom of the earth,” as also of Ungnad’s proposed [a-di pa]-a-ti, “to the ends” (Ungnad-Gressmann, Gilgamesch-Epos, p. 6, note), or a reading di-ma-a-ti, “pillars.” The first line of the Assyrian version would now read šá nak-ba i-mu-ru [dGis-gi(n)-maš i-ik-ka]-di ma-a-ti, i.e., “The one who saw everything, Gilgamesh the strong one (?) of the land.” We may at all events be quite certain that the name of the hero occurred in the first line and that he was described by some epithet indicating his superior position. Lines 229–235 are again an address of Gilgamesh to the sun-god, after having received a favorable “oracle” from the god (line 222). The hero promises to honor and to celebrate the god, by erecting thrones for him. Lines 237–244 describe the arming of the hero by the “master” craftsman. In addition to the pašu and paṭru, the bow (?) and quiver are given to him. Line 249 is paralleled in the new fragment of the Assyrian version published by King in PSBA 1914, page 66 (col. 1, 2), except that this fragment adds gi-mir to e-mu-ḳi-ka. Lines 251–252 correspond to column 1, 6–8, of King’s fragment, with interesting variations “battle” and “fight” instead of “way” and “road,” which show that in the interval between the old Babylonian and the Assyrian version, the real reason why Enkidu should lead the way, namely, because he knows the country in which Ḫuwawa dwells (lines 252–253), was supplemented by describing Enkidu also as being more experienced in battle than Gilgamesh. Line 254. I am unable to furnish a satisfactory rendering for this line, owing to the uncertainty of the word at the end. Can it [101]be “his household,” from the stem which in Hebrew gives us מִשְׁפָּחָה “family?” Line 255. Is paralleled by col. 1, 4, of King’s new fragment. The episode of Gišh and Enkidu proceeding to Ninsun, the mother of Gish, to obtain her counsel, which follows in King’s fragment, appears to have been omitted in the old Babylonian version. Such an elaboration of the tale is exactly what we should expect as it passed down the ages. Line 257. Our text shows that irnittu (lines 257, 264, 265) means primarily “endeavor,” and then success in one’s endeavor, or “triumph.” Lines 266–270. Do not appear to refer to rites performed after a victory, as might at a first glance appear, but merely voice the hope that Gišh will completely take possession of Ḫuwawa’s territory, so as to wash up after the fight in Ḫuwawa’s own stream; and the hope is also expressed that he may find pure water in Ḫuwawa’s land in abundance, to offer a libation to Šhamašh. Line 275. On šú-pa-as-su = šupat-su, see above, to l. 115. [Note on Sabitum (above, p. 11) In a communication before the Oriental Club of Philadelphia (Feb. 10, 1920), Prof. Haupt made the suggestion that sa-bi-tum (or tu), hitherto regarded as a proper name, is an epithet describing the woman who dwells at the seashore which Gilgamesh in the course of his wanderings reaches, as an “innkeeper”. It is noticeable that the term always appears without the determinative placed before proper names; and since in the old Babylonian version (so far as preserved) and in the Assyrian version, the determinative is invariably used, its consistent absence in the case of sabitum (Assyrian Version, Tablet X, 1, 1, 10, 15, 20; 2, 15–16 [sa-bit]; Meissner fragment col. 2, 11–12) speaks in favor of Professor Haupt’s suggestion. The meaning “innkeeper”, while not as yet found in Babylonian-Assyrian literature is most plausible, since we have sabū as a general name for ’drink’, though originally designating perhaps more specifically sesame wine (Muss-Arnolt, Assyrian Dictionary, p. 745b) or distilled brandy, according to Prof. Haupt. Similarly, in the Aramaic dialects, sebha is used for “to drink” and in the Pael to “furnish drink”. Muss-Arnolt in [102]his Assyrian Dictionary, 746b, has also recognized that sabitum was originally an epithet and compares the Aramaic sebhoyâthâ(p1) “barmaids”. In view of the bad reputation of inns in ancient Babylonia as brothels, it would be natural for an epithet like sabitum to become the equivalent to “public” women, just as the inn was a “public” house. Sabitum would, therefore, have the same force as šamḫatu (the “harlot”), used in the Gilgamesh Epic by the side of ḫarimtu “woman” (see the note to line 46 of Pennsylvania Tablet). The Sumerian term for the female innkeeper is Sal Geštinna “the woman of the wine,” known to us from the Hammurabi Code §§108–111. The bad reputation of inns is confirmed by these statutes, for the house of the Sal Geštinna is a gathering place for outlaws. The punishment of a female devotee who enters the “house of a wine woman” (bît Sal Geštinna §110) is death. It was not “prohibition” that prompted so severe a punishment, but the recognition of the purpose for which a devotee would enter such a house of ill repute. The speech of the sabitum or innkeeper to Gilgamesh (above, p. 12) was, therefore, an invitation to stay with her, instead of seeking for life elsewhere. Viewed as coming from a “public woman” the address becomes significant. The invitation would be parallel to the temptation offered by the ḫarimtu in the first tablet of the Enkidu, and to which Enkidu succumbs. The incident in the tablet would, therefore, form a parallel in the adventures of Gilgamesh to the one that originally belonged to the Enkidu cycle. Finally, it is quite possible that sabitum is actually the Akkadian equivalent of the Sumerian Sal Geštinna, though naturally until this equation is confirmed by a syllabary or by other direct evidence, it remains a conjecture. See now also Albright’s remarks on Sabitum in the A. J. S. L. 36, pp. 269 seq.] [103] 1 Scribal error for an. 2 Text apparently di. 3 Hardly ul. 4 Omitted by scribe. 5 Kišti omitted by scribe. 6 I.e., at night to thee, may Lugal-banda, etc. Corrections to the Text of Langdon’s Edition of the Pennsylvania Tablet.1 Column 1. 5. Read it-lu-tim (“heroes”) instead of id-da-tim (“omens”). 6. Read ka-ka-bu instead of ka-ka-’a. This disposes of Langdon’s note 2 on p. 211. 9 Read ú-ni-iš-šú-ma, “I became weak” (from enêšu, “weak”) instead of ilam iš-šú-ma, “He bore a net”(!). This disposes of Langdon’s note 5 on page 211. 10. Read Urukki instead of ad-ki. Langdon’s note 7 is wrong. 12. Langdon’s note 8 is wrong. ú-um-mid-ma pu-ti does not mean “he attained my front.” 14. Read ab-ba-la-áš-šú instead of at-ba-la-áš-šú. 15. Read mu-di-a-at instead of mu-u-da-a-at. 20. Read ta-ḫa-du instead of an impossible [sa]-ah-ḫa-ta—two mistakes in one word. Supply kima Sal before taḫadu. 22. Read áš-šú instead of šú; and at the end of the line read [tu-ut]-tu-ú-ma instead of šú-ú-zu. 23. Read ta-tar-ra-[as-su]. 24. Read [uš]-ti-nim-ma instead of [iš]-ti-lam-ma. 28. Read at the beginning šá instead of ina. 29. Langdon’s text and transliteration of the first word do not tally. Read ḫa-aṣ-ṣi-nu, just as in line 31. 32. Read aḫ-ta-du (“I rejoiced”) instead of aḫ-ta-ta. Column 2. 4. Read at the end of the line di-da-šá(?) ip-tí-[e] instead of Di-?-al-lu-un (!). 5. Supply dEn-ki-dū at the beginning. Traces point to this reading. 19. Read [gi]-it-ma-[lu] after dGiš, as suggested by the Assyrian version, Tablet I, 4, 38, where emûḳu (“strength”) replaces nepištu of our text. 20. Read at-[ta kima Sal ta-ḫa]-bu-[ub]-šú. 21. Read ta-[ra-am-šú ki-ma]. [104] 23. Read as one word ma-a-ag-ri-i-im (“accursed”), spelled in characteristic Hammurabi fashion, instead of dividing into two words ma-a-ak and ri-i-im, as Langdon does, who suggests as a translation “unto the place yonder(?) of the shepherd”(!). 24. Read im-ta-ḫar instead of im-ta-gar. 32. Supply ili(?) after ki-ma. 33. Read šá-ri-i-im as one word. 35. Read i-na [áš]-ri-šú [im]-ḫu-ru. 36. Traces at beginning point to either ù or ki (= itti). Restoration of lines 36–39 (perhaps to be distributed into five lines) on the basis of the Assyrian version, Tablet I, 4, 2–5. Column 3. 14. Read Kàš (= šikaram, “wine”) ši-ti, “drink,” as in line 17, instead of bi-iš-ti, which leads Langdon to render this perfectly simple line “of the conditions and the fate of the land”(!). 21. Read it-tam-ru instead of it-ta-bir-ru. 22. Supply [lùŠú]-I. 29. Read ú-gi-ir-ri from garû (“attack), instead of separating into ú and gi-ir-ri, as Langdon does, who translates “and the lion.” The sign used can never stand for the copula! Nor is girru, “lion!” 30. Read Síbmeš, “shepherds,” instead of šab-[ši]-eš! 31. šib-ba-ri is not “mountain goat,” nor can ut-tap-pi-iš mean “capture.” The first word means “dagger,” and the second “he drew out.” 33. Read it-ti-[lu] na-ki-[di-e], instead of itti immer nakie which yields no sense. Langdon’s rendering, even on the basis of his reading of the line, is a grammatical monstrosity. 35. Read giš instead of wa. 37. Read perhaps a-na [na-ki-di-e i]- za-ak-ki-ir. Column 4. 4. The first sign is clearly iz, not ta, as Langdon has it in note 1 on page 216. 9. The fourth sign is su, not šú. 10. Separate e-eš (“why”) from the following. Read ta-ḫi-[il], followed, perhaps, by la. The last sign is not certain; it may be ma. [105] 11. Read lim-nu instead of mi-nu. In the same line read a-la-ku ma-na-aḫ-[ti]-ka instead of a-la-ku-zu(!) na-aḫ … ma, which, naturally, Langdon cannot translate. 16. Read e-lu-tim instead of pa-a-ta-tim. The first sign of the line, tu, is not certain, because apparently written over an erasure. The second sign may be a. Some one has scratched the tablet at this point. 18. Read uk-la-at âli (?) instead of ug-ad-ad-lil, which gives no possible sense! Column 5. 2. Read [wa]-ar-ki-šú. 8. Read i-ta-wa-a instead of i-ta-me-a. The word pi-it-tam belongs to line 9! The sign pi is unmistakable. This disposes of note 1 on p. 218. 9. Read Mi = ṣalmu, “image.” This disposes of Langdon’s note 2 on page 218. Of six notes on this page, four are wrong. 11. The first sign appears to be si and the second ma. At the end we are perhaps to supply [šá-ki-i pu]-uk-ku-ul, on the basis of the Assyrian version, Tablet IV, 2, 45, šá-ki-i pu-[uk-ku-ul]. 12. Traces at end of line suggest i-pa(?)-ka-du. 13. Read i-[na mâti da-an e-mu]-ki i-wa. 18. Read ur-šá-nu instead of ip-šá-nu. 19. Read i-šá-ru instead of i-tu-ru. 24. The reading it-ti after dGiš is suggested by the traces. 25. Read in-ni-[ib-bi-it] at the end of the line. 28. Read ip-ta-ra-[aṣ a-la]-ak-tam at the end of the line, as in the Assyrian version, Tablet IV, 2, 37. 30. The conjectural restoration is based on the Assyrian version, Tablet IV, 2, 36. Column 6. 3. Read i-na ṣi-ri-[šú]. 5. Supply [il-li-ik]. 21. Langdon’s text has a superfluous ga. 22. Read uz-za-šú, “his anger,” instead of uṣ-ṣa-šú, “his javelin” (!). 23. Read i-ni-iḫ i-ra-as-su, i.e., “his breast was quieted,” in the sense of “his anger was appeased.” 31. Read ri-eš-ka instead of ri-eš-su. [106] In general, it should be noted that the indications of the number of lines missing at the bottom of columns 1–3 and at the top of columns 4–6 as given by Langdon are misleading. Nor should he have drawn any lines at the bottom of columns 1–3 as though the tablet were complete. Besides in very many cases the space indications of what is missing within a line are inaccurate. Dr. Langdon also omitted to copy the statement on the edge: 4 šú-ši, i.e., “240 lines;” and in the colophon he mistranslates šú-tu-ur, “written,” as though from šaṭâru, “write,” whereas the form is the permansive III, 1, of atâru, “to be in excess of.” The sign tu never has the value ṭu! In all, Langdon has misread the text or mistransliterated it in over forty places, and of the 204 preserved lines he has mistranslated about one-half. 1 The enumeration here is according to Langdon’s edition. Plates Plate I. The Yale Tablet. Plate II. The Yale Tablet. Plate III. The Yale Tablet. Plate IV. The Yale Tablet. Plate V. The Yale Tablet. Plate VI. The Yale Tablet. Plate VII. The Yale Tablet.

      Compared to the other versions focusing on the epic of Gilgamesh, this version looks more into Gilgamesh's cure for immortality after Enkidu's death. The "us" in this instance would be Gilgamesh and his search for a cure while the "them" would be the enemies which are trying stop him which include the forces he come along. The text is able to create this distinction by describing Gilgamesh as the main character as the one who is need of a cure because struggles to come to terms that he will die one day. Not to mention, Enkidu as a being was able to turn Gilgamesh into a noble figure who used his power for good turning him into a more likeable figure which is why the reader also roots for him to find a cure. Gilgamesh as a figure shows that in his time period, males were the ones who were seen as leaders who have strength because the other females in all versions of the text do not carry dynamic roles that showcase their personality or even their endearing qualities. There are more political and nationalistic themes compared to the Sumerian versions which illustrate how linguistics and language can play a role in how a culture might be perceived. By using the strong characteristics of Gilgamesh, the text is ultimately able to show the civilization of Uruk and create a sense of identity as a result. CC BY Ajey Sasimugunthan (contact)

  3. Apr 2024
    1. Author Response

      The following is the authors’ response to the original reviews.

      We would like to extend our gratitude to the reviewers for their meticulous analysis and constructive feedback on our manuscript. We have revised our paper based on the suggestions regarding supporting literature and the theory behind CAPs along with detailed insights regarding our methods. Their suggestions have been extremely useful in strengthening the clarity and rigor of our manuscript.

      Reviewer #1 (Recommendations For The Authors):

      (1) There are no obvious problems with this paper and it is relatively straightforward. There are some challenges that I would like to suggest. These variants have multiple mutations, so it would be interesting if you could drill down to find out which mutation is the most important for the collective changes reported here. I would like to see a sequence alignment of these variants, perhaps in the supplemental material, just to get some indication of the extent of mutations involved.

      Finding the most important mutation within a set is a tricky question, as each mutation changes the way future mutations will affect function due to epistasis. Indeed, this is what we aim to explore in this work. To illustrate this point, we included a new supplementary figure S5A. Three critical mutations that emerged quickly, and were frequently observed in other dominant variants, were S477N, T478K, and N501Y. Thus, we computed the EpiScore values of these three mutations, with several critical residues contributing to hACE2 binding. The EpiScore distribution indicates that residues 477, 478, and 501 have strong epistatic (i.e., non-additive) interactions, as indicated by EpiScore values above 2.0.

      To further investigate these epistatic interactions, we first conducted MD simulations and computed the DFI profile of these three single mutants. We analyzed how different the DFI scores of the hACE2 binding interface residues of the RBD are, across three single mutants with Omicron, Delta, and Omicron XBB variants (Fig S5B). Fig S5B shows how mutations at these particular sites affect the binding interface DFI in various backgrounds, as the three mutations are also observed in the Omicron, XBB, and XBB 1.5 variants. If the difference in the DFI profile of the mutant and the given variant is close to 0, then we could safely state that this mutation affected the variant the most. However, what we observe is quite the opposite: the DFI profile of the mutation is significantly different in different variant backgrounds. While these mutations may change overall behavior, their individual contributions to overall function are more difficult to pin down because overall function is dependent on the non-additive interactions between many different residues.

      Author response image 1.

      (A) Three critical mutations that emerged quickly, and were frequently observed in other dominant variants, were S477N, T478K, and N501Y. EpiScores of sites 477, 478, and 501 with one another are shown with k = the binding interface of the open chain. These residues are highly epistatic, producing higher responses than expected when perturbed together. (B) The difference in the dynamic flexibility profiles between the single mutants and the most common variants for the hACE2 binding residues of the RBD. DFI profiles exhibit significant variation from zero, and also show different flexibility in each background variant, highlighting the critical non-additive interactions of the other mutation in the given background variant. Thus, these three critical mutations, impacting binding affinity, do not solely contribute to the binding. There are epistatic interactions with the other mutations in VOCs that shape the dynamics of the binding interface to modulate binding affinity with hACE2.

      As we discussed above, while the epistatic interactions are crucial and the collective impact of the mutations shape the mutational landscape of the spike protein, we would like note that mutation S486P is one of the critical mutations we identify, modulating both antibody and hACE2 binding and our analysis reveals the strong non-additive interactions with the other mutational sites. This mutational site appears in both XBB1.5 and earlier Omicron strains which highlights its importance in functional evolution of the spike protein. CAPs 346R, 486F, and 498Q also may be important, as they have a high EpiScore, indicating critical epistatic interaction with many mutation sites.

      Regarding to the suggestion about presenting the alignment of the different variants, we have attached a mutation table, highlighting the mutated residues for each strain compared to the reference sequence as supplemental Figure S1 along with the full alignment file.

      (2) Also, I am wondering if it would be possible to insert some of these flexibilities and their correlations directly into the elastic network models to enable a simpler interpretation of these results. I realize this is beyond the scope of the present work, but such an effort might help in understanding these relatively complex effects.

      This is great suggestion. A similar analysis has been performed for different proteins by Mcleash (See doi: 10.1016/j.bpj.2015.08.009) by modulating the spring constants of specific position to alter specific flexibility and evaluate change in elastic free energy to identify critical mutation (in particular, allosteric mutation) sites. We will be happy to pursue this as future work.

      Minor

      (3) 1 typo on line 443 - should be binding instead of biding.

      Fixed, thanks for spotting that.

      (4) The two shades of blue in Fig. 4B were not distinguishable in my version.

      To fix this, we have changed the overlapping residues between Delta and Omicron to a higher contrast shade of blue.

      (5) Compensatory is often used in an entirely different way - additional mutations that help to recover native function in the presence of a deleterious mutation.

      Although our previous study (Ose et al. 2022, Biophysical Journal) shows that compensatory mutations were generally additive, the two ideas are not one and the same. We thank the reviewer for pointing this out. Therefore, to clarify, we have now described our results in terms of dynamic additivity, rather than compensation.

      Reviewer #2 (Recommendations For The Authors):

      (1) The authors note that the identified CAPs overlap with those of others (Cagliani et al. 2020; Singh and Yi 2021; Starr, Zepeda, et al. 2022). In itself, this merits a deeper discussion and explicit indication of which positions are not identified. However, there is one point that I believe may represent a fundamental flaw in this study in that the calculation of EP from the alignment of S proteins ignores entirely the differences in the interacting interface with which S for different coronaviruses in the alignment interact in the different receptors in each host species. This may be the reason why so many "CAPs" are in the RBD. The authors should at the very least make a convincing case of why they are not simply detecting constraints imposed by the different interacting partners, at least in the case of positions within the RBD interface with ACE2. Another point that the authors should discuss is that ACE2 is not the only receptor that facilitates infection, TMPRSS2 and possibly others have been identified as well. The results should be discussed in light of this.

      To begin with, we have now explicitly noted (on line 135) that “sites 478, 486, 498, and 681 have already been implicated in SARS-CoV-2 evolution, leaving the remaining 11 CAPs as undiscovered candidate sites for adaptation.” Evolutionary analyses are done using orthologous protein sequences, so there is no way to integrate information on different receptors in each host species in the calculation of EPs. However, we appreciate that the preponderance of CAPs in the RBD is likely due to different binding environments. We have added the following text (on line 83) to clarify our point: “Adaptation in this case means a virus which can successfully infect human hosts. As CAPs are unexpected polymorphisms under neutral theory, their existence implies a non-neutral effect. This can come in the form of functional changes (Liu et al. 2016) or compensation for functional changes (Ose et al. 2022). Therefore, we suspect that these CAPs, being unexpected changes from coronaviruses across other host species with different binding substrates, may be partially responsible for the functional change of allowing human infection.” This hypothesis is supported by the overlap of CAPs we identified with the positions identified in other studies (e.g., 478, 486, 498, and 681). Binding to TMPRSS2 and other substrates are also covered by this analysis as it is a measure of overall evolutionary fitness, rather than binding to any specific substrate. Our paper does focus on discussing hACE2 binding and mentions furin cleavage, but indeed lacks discussion on the role of TMPRSS2. We have added the following text to line 157: “Another host cell protease, TMPRSS2, facilitates viral attachment to the surface of target cells upon binding either to sites Arg815/Ser816, or Arg685/Ser686 which overlaps with the furin cleavage site 676-689, further emphasizing the importance of this area (Hoffmann et al. 2020b; Fraser et al. 2022).”

      (2) Turning now to the computational methods utilized to study dynamics, I have serious reservations about the novelty of the results as well as the validity of the methodology. First of all, the authors mention the work of Teruel et al. (PLOS Comp Bio 2021) in an extremely superficial fashion and do not mention at all a second manuscript by Teruel et al. (Biorxiv 2021.12.14.472622 (2021)). However, the work by Teruel et al. identifies positions and specific mutations that affect the dynamics of S and the evolution of the SARS-CoV-2 virus in light of immune escape, ACE2 binding, and open and closed state dynamics. The specific differences in approach should be noted but the results specifically should be compared. This omission is evident throughout the manuscript. Several other groups have also published on the use of nomal-mode analysis methods to understand the Spike protein, among them Verkhivker et al., Zhou et al., Majumder et al., etc.

      Thank you for your suggestions. Upon further examination of the listed papers, we have added citations to other groups employing similar methods. However, it's worth noting that the results of Teruel et al.'s studies are generally not directly comparable to our own. Particularly, they examine specific individual mutations and overall dynamical signatures associated with them, whereas our results are always considered in the context of epistasis and joint effects with CAPs, and all mutations belong to the common variants. Although important mutations may be highlighted in both cases, it is for very different reasons. Nevertheless, we provide a more detailed mention of the results of both studies. See lines 178, 255, and 393.

      (3) The last concern that I have is with respect to the methodology. The dynamic couplings and the derived index (DCI) are entirely based on the use of the elastic network model presented which is strictly sequence-agnostic. Only C-alpha positions are taken into consideration and no information about the side-chain is considered in any manner. Of course, the specific sequence of a protein will affect the unique placement of C-alpha atoms (i.e., mutations affect structure), therefore even ANM or ENM can to some extent predict the effect of mutations in as much as these have an effect on the structure, either experimentally determined or correctly and even incorrectly modelled. However, such an approach needs to be discussed in far deeper detail when it comes to positions on the surface of a protein such that the reader can gauge if the observed effects are the result of modelling errors.

      We would like to clarify that most of our results do not involve simulations of different variants, but rather how characteristic mutation sites for those variants contribute to overall dynamics. For the full spike, we operate on only two simulations: open and closed. When we do analyze different variants, starting on line 438, the observed difference does not come from the structure, but from the covariance matrix obtained from molecular dynamics (MD) simulations, which are sensitive to single amino acid changes.

      Reviewer #3 (Recommendations For The Authors):

      (1) On line 99 there is a misspelling, 'withing'.

      It has been fixed. Thanks for spotting that.

      (2) Some graphical suggestions to make the figures easier to read:

      In Figure 1C, a labeled circle around the important sites, the receptor binding domain, and the Furin cleavage site, would help the reader orient themselves. Moreover, it would make clear which CAPs are NOT in the noteworthy sites described in the text.

      Good idea. We have added transparent spheres and labels to show hACE2 binding sites and Furin cleavage sites.

      In Figure 2C the colors are a bit low contrast; moreover, there are multiple text sizes on the same figure which should perhaps be avoided to ensure legibility.

      We have made yellow brighter and standardized font sizes.

      Figure 3 is a bit dry, perhaps indicating in which bins the 'interesting' sites could be informative.

      Thank you for the suggestion, but the overall goal of Figure 3 is to illustrate that the mutational landscape is governed by the equilibrium dynamics in which flexible sites undergo more mutations during the evolution of the CoV2 spike protein. Therefore, adding additional positional information may complicate our message.

      Figure 4, the previous suggestions about readability apply.

      We ensured same sized text and higher contrast colors.

      Figure 5B, the residue labels are too small.

      We increased the font size of the residue labels.

      In Figure 8 maybe adding Delta to let the reader orient themselves would be helpful to the discussion.

      Unfortunately, there is no single work that has experimentally quantified binding affinities towards hACE2 for all the variants. When we conducted the same analysis for the Delta variant in Figure 8, the experimental values were obtained from a different source (doi: 10.1016/j.cell.2022.01.001) and the values were significantly different from the experimental work we used for Omicron (Yue et al. 2023). When we could adjust based on the difference in experimentally measured binding affinity values of the original Wuhan strain in these two separate studies, we observed a similar correlation, as seen below. However, we think this might not be a proper representation. Therefore, we chose to keep the original figure.

      Author response image 2.

      The %DFI calculations for variants Delta, Omicron, XBB, and XBB 1.5. (A) %DFI profile of the variants are plotted in the same panel. The grey shaded areas and dashed lines indicate the ACE2 binding regions, whereas the red dashed lines show the antibody binding residues. (B) The sum of %DFI values of RBD-hACE2 interface residues. The trend of total %DFI with the log of Kd values overlaps with the one seen with the experiments. (C) The RBD antibody binding residues are used to calculate the sum of %DFI. The ranking captured with the total %DFI agrees with the susceptibility fold reduction values from the experiments.

      (3) Replicas of the MD simulations would make the conclusions stronger in my opinion.

      We ran a 1µs long simulation and performed convergence analysis for the MD simulations using the prior work (Sawle L, Ghosh K. 2016.) More importantly, we also evaluated the statistical significance of computed DFI values as explained in detail below (Please see the answer to question 3 of Reviewer #3 (Public Review):)

      Reviewer #3 (Public Review):

      (1) A longer discussion of how the 19 orthologous coronavirus sequences were chosen would be helpful, as the rest of the paper hinges on this initial choice.

      The following explanation has been added on line 114: EP scores of the amino acid variants of the S protein were obtained using a Maximum Likelihood phylogeny (Kumar et al. 2018) built from 19 orthologous coronavirus sequences. Sequences were selected by examining available non-human sequences with a sequence identity of 70% or above to the human SARS CoV-2’s S protein sequence. This cutoff allows for divergence over evolutionary history such that each amino acid position had ample time to experience purifying selection, whilst limiting ourselves to closely related coronaviruses. (Figure 1A).

      (2) The 'reasonable similarity' with previously published data is not well defined, nor there was any comment about some of the residues analyzed (namely 417-484). We have revised this part of the manuscript and add to the revised version.

      We removed the line about reasonable similarity as it was vague, added a line about residues 417-484, and revised the text accordingly, starting on line 354.

      (3) There seem to be no replicas of the MD simulations, nor a discussion of the convergence of these simulations. A more detailed description of the equilibration and production schemes used in MD would be helpful. Moreover, there is no discussion of how the equilibration procedure is evaluated, in particular for non-experts this would be helpful in judging the reliability of the procedure.

      We opted for a single, extended equilibrium simulation to comprehensively explore the longterm behavior of the system. Given the specific nature of our investigation and resource constraints, a well-converged, prolonged simulation was deemed a practical and scientifically valid approach, providing a thorough understanding of the system's dynamics. (doi: 10.33011/livecoms.1.1.5957, https://doi.org/10.1146/annurev-biophys-042910-155255 )

      We updated our methods section starting on line 605 with extended information about the MD simulations and the converge criteria for the equilibrium simulations. We also added a section that explains our analysis to check statistical significance of obtained DFI values.

  4. Mar 2024
    1. Author Response

      The following is the authors’ response to the original reviews.

      Response to reviewer’s comments

      Reviewer #1 (Public Review):

      In this study, the structural characteristics of plant AlaDC and SerDC were analyzed to understand the mechanism of functional differentiation, deepen the understanding of substrate specificity and catalytic activity evolution, and explore effective ways to improve the initial efficiency of theanine synthesis.

      On the basis of previous solid work, the authors successfully obtained the X-ray crystal structures of the precursors of theanine synthesis-CsAlaDC and AtSerDC, which are key proteins related to ethylamine synthesis, and found a unique zinc finger structure on these two crystal structures that are not found in other Group II PLP-dependent amino acid decarboxylases. Through a series of experiments, it is pointed out that this characteristic zinc finger motif may be the key to the folding of CsAlaDC and AtSerDC proteins, and this discovery is novel and prospective in the study of theine synthesis.

      In addition, the authors identified Phe106 of CsAlaDC and Tyr111 of AtSerDC as key sites of substrate specificity by comparing substrate binding regions and identified amino acids that inhibit catalytic activity through mutation screening based on protein structure. It was found that the catalytic activity of CsAlaDCL110F/P114A was 2.3 times higher than that of CsAlaDC. At the same time, CsAlaDC and AtSerDC substrate recognition key motifs were used to carry out evolutionary analysis of the protein sequences that are highly homologous to CsAlaDC in embryos, and 13 potential alanine decarboxylases were found, which laid a solid foundation for subsequent studies related to theanine synthesis.

      In general, this study has a solid foundation, the whole research idea is clear, the experimental design is reasonable, and the experimental results provide strong evidence for the author's point of view. Through a large number of experiments, the key links in the theanine synthesis pathway are deeply studied, and an effective way to improve the initial efficiency of theanine synthesis is found, and the molecular mechanism of this way is expounded. The whole study has good novelty and prospectivity, and sheds light on a new direction for the efficient industrial synthesis of theanine

      Response: Thank you very much for taking time to review this manuscript. We appreciate all your insightful comments and constructive suggestions.

      Reviewer #1 (Recommendations For The Authors):

      (1) If some test methods are not original, references or method basis should be indicated.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have added references for the enzymatic activity experiments performed to measure the synthesis of theanine in the revised manuscript.

      (2) The conclusion is a little lengthy, and the summary of the whole study is not well condensed.

      Response: Thank you very much for your valuable suggestions. We have refined the conclusion in the revised manuscript, and it is as follows:

      In conclusion, our structural and functional analyses have significantly advanced understanding of the substrate-specific activities of alanine and serine decarboxylases, typified by CsAlaDC and AtSerDC. Critical amino acid residues responsible for substrate selection were identified—Tyr111 in AtSerDC and Phe106 in CsAlaDC—highlighting pivotal roles in enzyme specificity. The engineered CsAlaDC mutant (L110F/P114A) not only displayed enhanced catalytic efficiency but also substantially improved L-theanine yield in a synthetic biosynthesis setup with PsGS or GMAS. Our research expanded the repertoire of potential alanine decarboxylases through the discovery of 13 homologous enzyme candidates across embryophytic species and uncovered a special motif present in serine protease-like proteins within Fabale, suggesting a potential divergence in substrate specificity and catalytic functions. These insights lay the groundwork for the development of industrial biocatalytic processes, promising to elevate the production of L-theanine and supporting innovation within the tea industry.

      Reviewer #2 (Public Review)

      Summary:

      The manuscript focuses on the comparison of two PLP-dependent enzyme classes that perform amino acyl decarboxylations. The goal of the work is to understand the substrate specificity and factors that influence the catalytic rate in an enzyme linked to theanine production in tea plants.

      Strengths:

      The work includes x-ray crystal structures of modest resolution of the enzymes of interest. These structures provide the basis for the design of mutagenesis experiments to test hypotheses about substrate specificity and the factors that control catalytic rate. These ideas are tested via mutagenesis and activity assays, in some cases both in vitro and in plants.

      Weaknesses:

      The manuscript could be more clear in explaining the contents of the x-ray structures and how the complexes studied relate to the reactant and product complexes. The structure and mechanism section would also be strengthened by including a diagram of the reaction mechanism and including context about reactivity. As it stands, much of the structural results section consists of lists of amino acids interacting with certain ligands without any explanation of why these interactions are important or the role they play in catalysis. The experiments testing the function of a novel Zn(II)-binding domain also have serious flaws. I don't think anything can be said at this point about the function of the Zn(II) due to a lack of key controls and problems with experimental design.

      Response: Thank you very much for your thoughtful comments and feedback on our manuscript. We are pleased to hear that the work's strengths, such as the X-ray crystal structures and the mutagenesis experiments tied to the catalytic rate and substrate specificity, align with the goals of our research.

      We recognize the areas identified for improvement and appreciate the suggestions provided. We have emphasized how we use the structural information obtained to infer the roles of key amino acid residues in the reaction. Additionally, we have added a diagram of the reaction mechanism in the Supplementary figure to provide clearer context on reactivity and improve the overall understanding of the catalytic process. Regarding the structural results section, we have included a discussion that contextualizes the list of amino acids and their interactions with the ligands by explaining their significance and roles in catalysis. We acknowledge the weaknesses you've pointed out in the experiments concerning the novel Zn(II)-binding domain, but we would like to clarify that the focus of our study was not primarily on the zinc structure. While we agree that there may be limitations in the experimental design and controls for the zinc binding domain, we believe that these flaws do not significantly impact the overall findings of the study. The experiment served as a preliminary exploration of the potential functionality of the domain, and further studies are required to fully understand its role and mechanism.

      Reviewer #2 (Recommendations For The Authors):

      (1) In addition to the points raised in the public review, it would be ideal to provide some context for the enzymatic characterization. Why are the differences in kinetic parameters for AlaDC and SerDC significant?

      Response: Thank you for your comments and suggestions. The Km values for CsAlaDC and SerDCs are comparable, suggesting similar substrate affinities. However, CsAlaDC exhibits a significantly lower Vmax compared to AtSerDC and CsSerDC. This discrepancy implies that CsAlaDC and SerDCs may differ in the rates at which they convert substrate to product when saturated with substrate. SerDCs may have a faster turnover rate, meaning they convert substrate to product and release the enzyme more quickly, resulting in a higher Vmax. Differences in the stability or correct folding of the enzymes under assay conditions can also affect their Vmax. If SerDCs are more stable, they might maintain their catalytic activity better at higher substrate concentrations, contributing to a higher Vmax. We have added these to the part of “Enzymatic properties of CsAlaDC, AtSerDC, and CsSerDC” in our revised manuscript.

      (2) Why is Phe106/Tyr111 pair critical for substrate specificity? Does the amino acid contact the side chain? It might be helpful to a reader to formulate a hypothesis for this interaction.

      Response: Thank you for the question and comments. We conducted a comparison between the active sites of CsAlaDC and AtSerDC and observed a distinct difference in only two amino acids: F106 in CsAlaDC and Y111 in AtSerDC. The remaining amino acids were found to be identical. Expanding on previous research concerning Group II PLP-dependent amino acid decarboxylases, it was postulated and subsequently confirmed that these specific amino acids play a crucial role in substrate recognition. However, since we lack the structure of the enzyme-substrate complex, we are unable to elucidate the precise interactions occurring between the substrate and the amino acids at this particular site based solely on structural information.

      (3) Line 55 - Define EA again.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have redefined “EA” as the abbreviation for ethylamine in the revised manuscript.

      (4) Line 58 - The meaning of "determined by the quality formation of tea" is not clear.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have modified it in the revised manuscript.

      (5) Line 65 - Missing words between "despite they".

      Response: Thank you very much for your careful reading of the manuscript. We have corrected it in the revised manuscript.

      (6) Line 67 - Need a reference for the statement about lower activity?

      Response: Thank you for the question and comments. We have provided the following reference to support this statement in the revised manuscript.

      Reference: Bai, P. et al. (2021) Biochemical characterization of specific Alanine Decarboxylase (ADC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis). BMC Biotechnol. 21,17.

      (7) Line 100-101 - The meaning of "its closer relationship was Dicots plants." is not clear.

      Response: We have revised the sentence in the revised manuscript, as follows: “Phylogenetic analysis indicated that CsAlaDC is homologous with SerDCs in Dicots plants.”

      (8) Line 139 - Missing a word between "as well as" and "of".

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

      (9) Line 142 - The usage of comprised here is not correct. It would be more correct to say "The overall architecture of CsAlaDC and AtSerDC is homodimeric with the two subunits...".

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

      (10) Line 148-149 - I didn't understand the statement about the "N-terminal structures" Are these structures obtained from protein samples that have a truncated N-terminus?

      Response: Group II PLP-dependent amino acid decarboxylases are comprised of three distinct structural domains: the N-terminal domain, the large domain, and the C-terminal domain. Each of these domains possesses unique structural features. Similarly, CsAlaDC and AtSerDC can also be classified into three structural domains based on their specific characteristics. To achieve more stable proteins for further experiments, we conducted truncation on both of these proteins. The truncated section pertains to a subsection of the N-terminal domain and is truncated from the protein's N-terminus.

      (11) Line 153 - Say "is composed of" instead of "composes of".

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

      (12) Line 156 - I didn't understand the statement about the cofactor binding process. What is the cofactor observed? And how can we say anything about the binding process from a single static structure of the enzyme? It might be better to say that the cofactor binding site is located at the subunit junction - but the identity of the cofactor still needs to be defined first.

      Response: Thank you for your comments and suggestions. The cofactor mentioned here is PLP. We aim to elucidate the binding state of PLP at the active site, excluding the binding process. The description has been revised in the revised manuscript.

      (13) Lines 157-158 - I didn't understand the conclusion about the roles of each monomer. In the images in Figure 3 - both monomers appear to bind PLP but the substrate is not present - so it's not clear how conclusions can be drawn about differential substrate binding in the two subunits.

      Response: Thank you very much for your careful reading and valuable suggestions. The main idea we want to convey is that this protein possesses two active sites. At each active site, the two monomers carry out distinct functions. Of course, our previous conclusion is inaccurate due to the non-existence of the substrate. So, we have made the necessary amendments in the revised manuscript.

      (14) Line 161 - I would say loop instead of ring.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected it in the revised manuscript.

      (15) Line 165 - Please provide some references for this statement. It would also be ideal to state the proximity of the Zn-binding motif to the active site or otherwise provide some information about the role of the motif based on its location.

      Response: Thank you for your comments and suggestions. We have provided the following references to support this statement in the revised manuscript.

      Author response image 1.

      (A) Structure of histidine decarboxylase. (B) Structure of glutamate decarboxylase.

      Reference:

      30 Komori, H. et al. (2012) Structural study reveals that Ser-354 determines substrate specificity on human Histidine Decarboxylase. J Biol Chem. 287, 29175-83.

      31 Huang, J. et al. (2018) Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis. Biochem Biophys Res Co. 503, 1703-1709

      In CsAlaDC, the zinc is positioned at a distance of 29.6 Å from the active center, whereas in AtSerDC, the zinc is situated 29 Å away from the active center. Hence, we hypothesize that this structure does not impact the enzyme's catalytic activity but might be correlated with its stability.

      (16) Lines 166-178 - This paragraph appears to be a list of all of the interactions between the protein, PLP, and the EA product. It would be ideal to provide some text to explain why these interactions are important and what we can learn from them.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have been conducting additional analysis on the functional roles of amino acid residues involved in the interaction between the active site and PLP. This analysis focuses on aiding PLP binding, determining its orientation, and understanding enzyme catalytic mechanisms. These details are mentioned in the revised manuscript.

      (17) Line 192 - Bond not bound.

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have made corrections in the revised manuscript.

      (18) Lines 201-207 - It would be ideal to verify that the inclusion of 5 mM DTT affects Zn binding. It's not clear to me that this reagent would necessarily disrupt Zn binding. Under certain circumstances, it could instead promote Zn association. For example, if the Cys ligands are oxidized initially but then become reduced? I don't think the current experiment really provides any insight into the role of the Zn.

      Response: Thank you for your valuable insights regarding the role of DTT and its potential effects on Zn binding in our experiments. The main function of DTT is to protect or restore the reduced state of proteins and other biological molecules, particularly by disrupting the crosslinking formed by thiol (-SH) groups and disulfide bonds to maintain the function and structure of proteins. Therefore, the reason for DTT's inhibition of enzyme activity is unknown, and we cannot provide a reasonable explanation for this phenomenon. As a result, we have removed the section discussing the inhibition of enzyme activity by DTT in our revised manuscript.

      Reviewer #3 (Public Review):

      In the manuscript titled "Structure and Evolution of Alanine/Serine Decarboxylases and the Engineering of Theanine Production," Wang et al. solved and compared the crystal structures of Alanine Decarboxylase (AlaDC) from Camellia sinensis and Serine Decarboxylase (SerDC) from Arabidopsis thaliana. Based on this structural information, the authors conducted both in vitro and in vivo functional studies to compare enzyme activities using site-directed mutagenesis and subsequent evolutionary analyses. This research has the potential to enhance our understanding of amino acid decarboxylase evolution and the biosynthetic pathway of the plant-specialized metabolite theanine, as well as to further its potential applications in the tea industry. Response: Thank you very much for taking the time to review this manuscript. We appreciate all your insightful comments.

      Reviewer #3 (Recommendations For The Authors):

      Page 6, Figure 2, Page 23 (Methods)

      "The supernatants were purified with a Ni-Agarose resin column followed by size-exclusion chromatography."

      What kind of SEC column did the authors use? Can the authors provide the SEC elution profile comparison results and size standard curve?

      Response: We use a Superdex 200 (Hiload 16/600) column for size exclusion chromatography. The comparison results of SEC elution profiles for AtSerDC and CsAlaDC, along with the standard curve of SEC column, are presented below.

      Author response image 2.

      (A) Comparison of elution profiles of CsAlaDC and AtSerDC. (B) Elution profile of Blue Dextron 2000. (C) Elution profile of mixed protein (Aldolase, 158000 Da,71.765ml; Conalbumin, 75000 Da,79.391ml; Ovalbumin, 44000 Da,83.767ml; Carbonic anhydrase, 29000 Da,90.019ml; Ribonuclease A, 13700 Da,98.145ml). (D) Size standard curves of Superdex 200 (Hiload 16/600) column.

      Page 6 & Page 24 (Methods)

      "The 100 μL reaction mixture, containing 20 mM substrate (Ala or Ser), 100 mM potassium phosphate, 0.1 mM PLP, and 0.025 mM purified enzyme, was prepared and incubated at standard conditions (45 ℃ and pH 8.0 for CsAlaDC, 40 ℃ and pH 8.0 for AtSerDC for 30 min)."

      (1) The enzymatic activities of CsAldDC and AtSerDC were measured at two different temperatures (45 and 40 ℃, but their activities were directly compared. Is there a reason for experimenting at different temperatures?

      Response: We determined that the optimal reaction temperature for AtSerDC is 40°C and for CsAlaDC is 45°C through our verification process. Consequently, all subsequent experiments were performed at these specific temperatures.

      Author response image 3.

      (A) Relative activity of CsAlaDC at different temperatures. (B) Relative activity of AtSerDC at different temperatures.

      (2) Enzyme activities were measured at temperatures above 40℃, which is not a physiologically relevant temperature and may affect the stability or activity of the proteins. At the very least, the authors should provide temperature-dependent protein stability data (e.g., CD spectra analysis) or, if possible, temperature-dependent enzyme activities, to show that their experimental conditions are suitable for studying the activities of these enzymes.

      Response: Thank you very much for your careful reading. We have already validated that the experimental temperature we used did not significantly affect the stability of the protein before experimenting. The results are shown in the figure below:

      Author response image 4.

      Place the two proteins individually into water baths set at temperatures of 25°C, 37°C, 45°C, 60°C, and 80°C for 15 minutes. Subsequently, carry out enzymatic reactions utilizing a standard reaction system, with untreated enzymes serving as the experimental control within the said system. The experimental results suggest that the temperature at which we experimented does not have a significant impact on the stability of the enzyme.

      (3) The authors used 20 mM of substrate. What are the physiological concentrations of alanine and serine typically found in plants?

      Response: The content of alanine in tea plant roots ranges from 0.28 to 4.18 mg/g DW (Yu et al., 2021; Cheng et al., 2017). Correspondingly, the physiological concentration of alanine is 3.14 mM to 46.92 mM, in tea plant roots. The content of serine in plants ranges from 0.014 to 17.6 mg/g DW (Kumar et al., 2017). Correspondingly, the physiological concentration of serine is 0.13 mM to 167.48 mM in plants. In this study, the substrate concentration of 20 mM was close to the actual concentrations of alanine and serine in plants.

      Yu, Y. et al. (2021) Glutamine synthetases play a vital role in high accumulation of theanine in tender shoots of albino tea germplasm "Huabai 1". J. Agric. Food Chem. 69 (46),13904-13915.

      Cheng, S. et al. (2017) Studies on the biochemical formation pathway of the amino acid L-theanine in tea (Camellia sinensis) and other plants.” J. Agric. Food Chem. 65 (33), 7210-7216.

      Kumar, V. et al. (2017) Differential distribution of amino acids in plants. Amino Acids. 49(5), 821-869.

      Pages 6-7 & Table 1

      (1) Use the correct notation for Km and Vmax. Also, the authors show kinetic parameters and use multiple units (e.g., mmol/L or mM for Km).

      Response: Thank you very much for your careful reading of the manuscript and valuable suggestions. We have corrected this in the revised manuscript.

      (2) When comparing the catalytic efficiency of enzymes, kcat/Km (or Vmax/Km) is generally used. The authors present a comparison of catalytic activity from results to conclusion. A clarification of what results are being compared is needed.

      Response: Thank you for your comments and suggestions. The catalytic activity is assessed by comparing reaction rates.

      Page 7 & Figure 3

      In Figure 3A, the authors describe the overall structure, but a simple explanation or labeling within the figure should be added.

      Response: Thank you very much for your suggestions, we have made modifications to Figure 3A as follows:

      Author response image 5.

      Crystal structures of CsAlaDC and AtSerDC. (A) Dimer structure of CsAlaDC. The color display of the N-terminal domain, large domain, and C-terminal domains of chain A is shown in light pink, khaki and sky blue, respectively. Chain B is shown in spring green. The PLP molecule is shown as a sphere model. The zinc finger structure at the C-terminus of CsAlaDC is indicated by the red box. The gray spheres represent zinc ions, while the red dotted line depicts the coordination bonds formed by zinc ions with cysteine and histidine.

      Figures 3F & 4A

      In these figures, the two structures are overlaid and compared, but the colors are very similar to see the differences. The authors should use a different color scheme.

      Response: Thank you very much for your suggestions, we have made modifications to the Figure 3F & 4A as follows:

      Author response image 6.

      (Figure 3F) The monomers of CsAlaDC and AtSerDC are superimposed. CsAlaDC is depicted in spring green, while AtSerDC is shown in plum. The conserved amino acid catalytic ring is indicated by the red box.

      (Figure 4A) Superposition of substrate binding pocket amino acid residues in CsAlaDC and AtSerDC. The amino acid residues of CsAlaDC are shown in spring green, the amino acid residues of AtSerDC are shown in plum, with the substrate specificity-related amino acid residue highlighted in a red ellipse.

      Pages 7 & 8

      Figures 3 and 4 do not include illustrations of what the authors describe in the text. The reader will not be able to understand the descriptions until they download and view the structures themselves. The authors should create additional figures to make it easier for readers to understand the structures.

      Response: Thank you very much for your suggestions, we have included supplementary figure 1 in the revised manuscript, which presents more elaborate structural depictions of the two proteins.

      Pages 9 & 10

      "This result suggested this Tyr is required for the catalytic activity of CsAlaDC and AtSerDC."

      The author's results are interesting, but it is recommended to perform the experiments in a specific order. First, experiments should determine whether mutagenesis affects the protein's stability (e.g., CD, as discussed earlier), and second, whether mutagenesis affects ligand binding (e.g., ITC, SPR, etc.), before describing how site-directed mutagenesis alters enzyme activity. In particular, the authors' hypothesis would be much more convincing if they could show that the ligand binding affinity is similar between WT and mutants.

      Response: Thank you for your insightful feedback on our manuscript, which we greatly appreciate. Your suggestion to methodically sequence the experiments provides a clear pathway to bolster the strength and conclusiveness of our results.

      We agree that it is crucial to first assess the stability of the mutant proteins, as changes therein could inadvertently affect catalytic activity. To this end, we have employed circular dichroism (CD) to study the potential structural alterations in the proteins induced by mutations. The experimental results are shown in the following figure:

      Author response image 7.

      (A) Circular Dichroism Spectra of CsAlaDC (WT). (B) Circular Dichroism Spectra of CsAlaDC (Y336F). (C) Circular Dichroism Spectra of CD of AtSerDC (WT). (D) Circular Dichroism Spectra of AtSerDC (Y341F).

      The experimental results indicate that the secondary structure of the mutant proteins remains unchanged, which means the mutations do not alter the protein's stability.

      The ligand PLP forms a Schiff base structure with the ε-amino group of a lysine residue in the protein, with maximum absorbance around 420-430 nm. Since we have already added PLP during the protein purification process, as long as the absorbance of mutant proteins and wild-type proteins is the same at 420-430 nm at equivalent concentrations, it indicates that the mutant proteins do not affect the binding of the ligand PLP. Therefore, we scanned the UV-visible absorption spectra of both the wild-type and mutant proteins, and the results are as presented in the following figure:

      Author response image 8.

      (A) UV-Visible Absorption Spectra of CsAlaDC (WT) compared to CsAlaDC (Y336F). (B) UV-Visible Absorption Spectra of AtSerDC (WT) compared to AtSerDC (Y341F).

      The mutant protein and the wild-type protein exhibit similar absorbance at 420-430 nm, indicating that the mutation does not affect the binding of PLP to the protein.

      The above experiments have confirmed that the mutations do not significantly affect the stability of the protein or the affinity for the ligand, so we can more confidently attribute changes in enzyme activity to the specific role of the tyrosine residue in question. We believe this comprehensive approach will substantiate our hypothesis and illustrate the necessity of this Tyr residue for the catalytic activity of CsAlaDC and AtSerDC enzymes.

      Figure 3

      In the 3D structure figure provided by the authors, the proposed reaction mechanism of the enzyme and the involved amino acids are not included. Can the authors add a supplementary figure with a schematic drawing that includes more information, such as distances?

      Response: Thank you for your valuable feedback on our manuscript. We completely agree that a schematic drawing with additional details, including distances, would enhance the clarity and understanding of the enzymatic mechanism. In response to your suggestion, we have added a supplementary figure 2 in the revised manuscript that accurately illustrates the proposed reaction pathway, highlighting the key amino acids involved.

      Page 10

      "The results showed that 5 mM L-DTT reduced the relative activity of CsAlaDC and AtSerDC to 22.0% and 35.2%, respectively"

      The authors primarily use relative activity to compare WT and mutants. Can the authors specify the exact experiments, units, and experimental conditions? Is it Vmax or catalytic efficiency? If so, under what specific experimental conditions?

      Response: Thank you for your attention and review of our research paper, we appreciate your suggestions and feedback. The experimental protocol employed to evaluate the influence of DTT on protein catalytic efficiency is outlined as follows:

      The 100 μL reaction mixture, containing 20 mM substrate (Ala or Ser), 100 mM potassium phosphate, 0.1 mM PLP, 5 mM L-DTT, and 0.025 mM purified enzyme, was prepared and incubated at standard conditions (45 °C and pH 8.0 for CsAlaDC for 5 min, 40 °C and pH 8.0 for AtSerDC for 2 min). DTT is absent as a control in the reaction system. Then the reaction was stopped with 20 μL of 10% trichloroacetic acid. The product was derivatized with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) and subjected to analysis by UPLC. All enzymatic assays were performed in triplicate.

      However, due to the unknown mechanism of DTT inhibition on protein activity, we have removed this part of the content in the revised manuscript.

      Pages 10-12

      The identification of 'Phe106 in CsAlaDC' and 'Tyr111 in AtSerDC,' along with the subsequent mutagenesis and enzymatic activity assays, is intriguing. However, the current manuscript lacks an explanation and discussion of the underlying reasons for these results. As previously mentioned, it would be helpful to gain insights and analysis from WT-ligand and mutant-ligand binding studies (e.g., ITC, SPR, etc.). Furthermore, the authors' analysis would be more convincing with accompanying structural analysis, such as steric hindrance analysis.

      Response: Thank you for your insightful comments and constructive feedback on our manuscript. We appreciate the interest you have expressed in the identification of 'Phe106 in CsAlaDC' and 'Tyr111 in AtSerDC' and their functional implications based on mutagenesis and enzymatic assays.

      In order to investigate the binding status of the mutant protein and the ligand PLP,we scanned the UV-visible absorption spectra of both the wild-type and mutant proteins, and the results are as presented in the following figure:

      Author response image 9.

      (A) UV-Visible Absorption Spectra of CsAlaDC (WT) compared to CsAlaDC (F106Y). (B) UV-Visible Absorption Spectra of AtSerDC (WT) compared to AtSerDC (Y111F).

      The mutant protein and the wild-type protein exhibit similar absorbance at 420-430 nm, indicating that the mutation does not affect the binding of PLP to the protein. Therefore, we can conclude that the change in activity of the mutant protein is caused by the substitution of the amino acid at that site, i.e., the amino acid at that site affects substrate specificity. By combining the structure of the two proteins, we can see that the Lys at position 111 of AtSerDC is a hydrophilic amino acid, which increases the hydrophilicity of the active site, and thus the substrate is the hydrophilic amino acid Ser. In contrast, the amino acid at the corresponding site in CsAlaDC is Phe, which, lacking a hydroxyl group compared to Lys, increases the hydrophobicity of the active site, making the substrate lean towards the hydrophobic amino acid Ala. We have added a discussion of the potential reasons for this result to the revised manuscript's discussion section.

      Page 5 & Figure 1B

      "As expected, CsSerDC was most closed to AtSerDC, which implies that they shared similar functions. However, CsAlaDC is relatively distant from CsSerDC."

      In Figure 1B, CsSerDC and AtSerDC are in different clades, and this figure does not show that the two enzymes are closest. To provide another quantitative comparison, please provide a matrix table showing amino acid sequence similarities as a supplemental table.

      Response: Many thanks for your constructive suggestion. We added a matrix table showing amino acid sequence similarities in the supplemental materials. The results showed that the similarity of amino acid sequences between CsSerDC and AtSerDC is 86.21%, which is higher than that between CsAlaDC and CsSerDC (84.92%). This data exactly supports the description of Figure 1B. We added the description of the amino acid sequence similarities analysis in the revised manuscript. The description of "As expected, CsSerDC was most closed to AtSerDC, which implies that they shared similar functions. " is not accurate enough, so we revised it to "As expected, CsSerDC was closer to AtSerDC, which implies that they shared similar functions.", in the revised manuscript.

      Page 5 & Figure 1C

      Figure 1C, which shows a multiple sequence alignment with the amino acid sequences of the 6 SerDCs and CsAlaDC, clearly shows the differences between the sequences of AlaDC and other SerDCs. However, the authors' hypothesis would be more convincing if they showed that this difference is also conserved in AlaDCs from other plants. Can the authors show a new multiple-sequence alignment by adding more amino acid sequences of other AlaDCs?

      Response: Thank you for your comments and suggestions. We aim to discover additional alanine decarboxylase. However, at present, the only experimentally confirmed alanine decarboxylase is CsAlaDC. No experimentally verified alanine decarboxylases have been found in other plant species.

      Figure 5A

      Figure 5A is missing the error bar.

      Response: Figure 5A serves as a preliminary screening for these mutants, without conducting repeated experiments. Subsequently, only the L110F and P114A mutants, which exhibited significantly improved activity, underwent further experimental verification to confirm their enhanced functionality.

  5. Nov 2023
    1. Author Response

      The following is the authors’ response to the original reviews.

      Thank you for your time and effort in handling and reviewing our manuscript. We have responded to all comments below.

      Reviewer #1 (Public Review):

      Martinez-Gutierrez and colleagues presented a timeline of important bacteria and archaea groups in the ocean and based on this they correlated the emergence of these microbes with GOE and NOE, the two most important geological events leading to the oxygen accumulation of the Earth. The whole study builds on molecular clock analysis, but unfortunately, the clock analysis contains important errors in the calibration information the study used, and is also oversimplified, leaving many alternative parameters that are known to affect the posterior age estimates untested. Therefore, the main conclusion that the oxygen availability and redox state of the ocean is the main driver of marine microbial diversification is not convincing.

      We do not conclude that “oxygen availability and redox state of the ocean is the main driver of marine microbial diversification”. Our conclusion is much more nuanced. We merely discuss our findings in light of the major oxygenation events and oxygen availability (among other things) given the important role this molecule has played in shaping the redox state of the ocean.

      Regarding the methodological concerns, to address them we have provided additional analyses to account for different clock models and calibration points.

      Basically, what the molecular clock does is to propagate the temporal information of the nodes with time calibrations to the remaining nodes of the phylogenetic tree. So, the first and the most important step is to set the time constraints appropriately. But four of the six calibrations used in this study are debatable and even wrong.

      (1) The record for biogenic methane at 3460 Ma is not reliable. The authors cited Ueno et al. 2006, but that study was based on carbon isotope, which is insufficient to demonstrate biogenicity, as mentioned by Alleon and Summons 2019.

      Thank you for pointing out the limitations of using the geochemical evidence of methane as calibrations. Indeed, several commentaries have suggested that the biotic and abiotic origin of the methane reported by Ueno et al. are equally plausible (Alleon and Summons, 2019; Lollar and McCollom, 2006), however; we used that calibration as a minimum for the presence of life on Earth, not methanogenesis. Despite the controversy regarding the origin of methane, there are other lines of evidence suggesting the presence of life around ~3.4 Ga. For example stromatolites from the Dresser Formation, Pilbara, Western Australia (Djokic et al., 2017; Walter et al., 1980; Buick and Dunlop, 1990), and more recently (Hickman-Lewis et al., 2022). To avoid confusion, we have added a more extended explanation for the use of that calibration and additional evidence of life around that time in Table 1 and lines 100-104.

      (2) Three calibrations at Aerobic Nitrososphaerales, Aerobic Marinimicrobia, and Nitrite oxidizing bacteria have the same problem - they are all assumed to have evolved after the GOE where the Earth started to accumulate oxygen in the atmosphere, so they were all capped at 2320 Ma. This is an important mistake and will significantly affect the age estimates because maximum constraint was used (maximum constraint has a much greater effect on age estimates and minimum constraint), and this was used in three nodes involving both Bacteria and Archaea. The main problem is that the authors ignored the numerous evidence showing that oxygen can be produced far before GOE by degradation of abiotically-produced abundant H2O2 by catalases equipped in many anaerobes, also produced by oxygenic cyanobacteria evolved at least 500 Ma earlier than the onset of GOE (2500 Ma), and even accumulated locally (oxygen oasis). It is well possible that aerobic microbes could have evolved in the Archaean.

      We appreciate the suggestion of assessing the validity of the calibrations used in our analyses. We initially evaluated the informative power of the priors used for the Bayesian molecular dating (Supplemental File 5), and found that the only calibration that lacked enough information for the purposes of our study was Ammonia Oxidizing Archaea (AOA). In contrast to previous evidence (Ren et al., 2019; Yang et al., 2021), we associate this finding to the potential earlier diversification of AOA. Due to the limitations of several of the calibrations used, we performed an additional molecular dating analysis on 1000 replicate trees using a Penalized Likelihood strategy. This analysis consisted in excluding the calibrations that assumed the presence of oxygen as a maximum constraint. Our analysis shows similar age estimates of the marine microbial clades regardless of the exclusion of these calibrations (Supplemental File 8; TreePL Priors set 2). Our findings thus suggest that the age estimates reported in our study are consistent regardless of whether or not the presence of oxygen is used to calibrate several nodes in the tree. We describe the results of this analysis in lines 490-499 and include estimates in Supplemental File 8. Our results are therefore robust regardless of the use of these somewhat controversial calibrations.

      Once the phylogenetic tree is appropriately calibrated with fossils and other time constraints, the next important step is to test different clock models and other factors that are known to significantly affect the posterior age estimates. For example, different genes vary in evolutionary history and evolutionary rate, which often give very different age estimates. So it is very important to demonstrate that these concerns are taken into account. These are done in many careful molecular dating studies but missing in this study.

      We agree that the selection of marker genes will have a profound impact on the final age estimates. First, it is important to understand that very few genes present in modern Bacteria and Archaea can be traced back to the Last Universal Common Ancestor, so there are very few genes to use for this purpose. Studies that focus on particular groups of Bacteria and Archaea may have larger selections of genes to choose from, but for our purposes there are only about ~40 different genes - mostly encoding for ribosomal proteins, RNA polymerase subunits, and tRNA synthetases - that can be use for this purpose (Creevey et al., 2011; Wu and Scott, 2012). In a previous study we have extensively benchmarked methods for the reconstruction of high-resolution phylogenetic trees of Bacteria and Archaea using these genes (Martinez-Gutierrez and Aylward, 2021). Our analyses demonstrated that some of these genes (mainly tRNA synthetases) have undergone ancient lateral gene transfer events and are not suitable for deep phylogenetics or molecular dating. In this previous study we also evaluated different sets of marker genes to examine which provide the most robust phylogenetic inference. We arrived at a set of ribosomal proteins and RNA polymerase subunits that performs best for phylogenetic reconstruction, and we have used that in the current study.

      Furthermore, we tested the role of molecular dating model selection on the final Bayesian estimates by running four independent chains under the models UGAM and CIR, respectively. Overall, the results did not vary substantially compared with the ages obtained using the log-normal model reported on our manuscript (Supplemental File 8). The additional results are described in lines 478-488 and shown in Supplemental File 8. The clades that showed more variation when using different Bayesian models were SAR86, SAR11, and Crown Cyanobacteria (Supplemental File 8). Despite observing some differences in the age estimates when using different molecular models, the conclusion that the different marine microbial clades presented in our study diversified during distinct periods of Earth’s history remains. Moreover, the main goal of our study is to provide a relative timeline of the diversification of abundant marine microbial clades without focusing on absolute dates.

      Reviewer #2 (Public Review):

      In this paper, Martinez-Gutierrez and colleagues present a dated, multidomain (= Archaea+Bacteria) phylogenetic tree, and use their analyses to directly compare the ages of various marine prokaryotic groups. They also perform ancestral gene content reconstruction using stochastic mapping to determine when particular types of genes evolved in marine groups.

      Overall, there are not very many papers that attempt to infer a dated tree of all prokaryotes, and this is a distinctive and up-to-date new contribution to that oeuvre. There are several particularly novel and interesting aspects - for example, using the GOE as a (soft) maximum age for certain groups of strictly aerobic Bacteria, and using gene content enrichment to try to understand why and how particular marine groups radiated.

      Thank you for your thorough evaluation and comments on our manuscript.

      Comments

      One overall feature of the results is that marine groups tend to be quite young, and there don't seem to be any modern marine groups that were in the ocean prior to the GOE. It might be interesting to study the evolution of the marine phenotype itself over time; presumably some of the earlier branches were marine? What was the criterion for picking out the major groups being discussed in the paper? My (limited) understanding is that the earliest prokaryotes, potentially including LUCA, LBCA and LACA, was likely marine, in the sense that there would not yet have been any land above sea level at such times. This might merit discussion in the paper. Might there have been earlier exclusively marine groups that went extinct at some point?

      Thank you for pointing this out - this is a very interesting idea.<br /> Firstly, the major marine lineages that we study here have largely already been defined in previous studies and are known to account for a large fraction of the total diversity and biomass of prokaryotes in the ocean. For example, Giovannoni and Stingl described most of these groups previously when discussing cosmopolitan and abundant marine lineages (Giovannoni and Stingl, 2005). The main criteria to select the marine clades studied here are 1) these groups have large impacts in the marine biogeochemical cycles and represent a large fraction of the microbial biomass in the open ocean, 2) they have an appropriate representation on genomic databases such that they can be confidently included in a phylogenetic tree, 3) the clades included can be confidently classified as being marine, in the sense that consequently the last common ancestor had a marine origin. This is explained in lines 83-86. We were primarily interested in lineages that encompassed a broad phylogenetic breadth, and we therefore did not include many groups that can be found in the ocean but are also readily isolated from a range of other environments (i.e., Pseudomonas spp., some Actinomycetes, etc.).

      We agree that some of the earlier microbial branches in the Tree of Life were likely marine. The study of the marine origin of LUCA, LBCA, LACA, although interesting, is out of the scope of our study, and our results cannot offer any direct evidence of their habitat. We have therefore sought to focus on the origins of extant marine lineages.

      What do the stochastic mapping analyses indicate about the respective ancestors of Gracilicutes and Terrabacteria? At least in the latter case, the original hypothesis for the group was that they possessed adaptations to life on land - which seems connected/relevant to the idea of radiating into the sea discussed here - so it might be interesting to discuss what your analyses say about that idea.

      Thank you for your recommendation to perform additional analysis regarding the characterization of the ancestor of the superphyla Gracilicutes and Terrabacteria. We agree that this analysis would be very interesting, but we wish to focus the manuscript primarily on the marine clades in question, and other supergroups are listed in Figure 2 mainly for context. However, we did check the results of the stochastic mapping analysis and we now report the list of genes predicted to be gained and lost at the ancestor of the Gracilicutes and Terrabacteria clades, however; it is out of the scope of this study.

      I very much appreciate that finding time calibrations for microbes is challenging, but I nonetheless have a couple of comments or concerns about the calibrations used here:

      The minimum age for LBCA and LACA (Nodes 1 and 2 in Fig. 1) was calibrated with the earliest evidence of biogenic methane ~3.4Ga. In the case of LACA, I suppose this reflects the view that LACA was a methanogen, which is certainly plausible although perhaps not established with certainty. However, I'm less clear about the logic of calibrating the minimum age of Bacteria using this evidence, as I am not aware that there is much evidence that LBCA was a methanogen. Perhaps the line of reasoning here could be stated more explicitly. An alternative, slightly younger minimum age for Bacteria could perhaps be obtained from isotope data ~3.2Ga consistent with Cyanobacteria (e.g., see https://pubmed.ncbi.nlm.nih.gov/30127539/).

      Thank you for pointing this out. We used the presence of methane as a minimum for life on Earth, not as a minimum for methanogenesis. Despite using this calibration as a minimum for the root of Bacteria and not having methanogenic representatives within this domain, there are independent lines of evidence that point to the presence of microbial life around the same time (~3.5 Ga, for example stromatolites from the Dresser Formation, Pilbara, Western Australia (~3.5 Ga) (Djokic et al., 2017; Walter et al., 1980; Buick and Dunlop, 1990), and more recently (Hickman-Lewis et al., 2022). We added a rationale for the use of the evidence of methane as a minimum age for life on Earth to the manuscript (Table 1 and 100104).

      I am also unclear about the rationale for setting the minimum age of the photosynthetic Cyanobacteria crown to the time of the GOE. Presumably, oxygen-generating photosynthesis evolved on the stem of (photosynthetic) Cyanobacteria, and it therefore seems possible that the GOE might have been initiated by these stem Cyanobacteria, with the crown radiating later? My confusion here might be a comprehension error on my part - it is possible that in fact one node "deeper" than the crown was being calibrated here, which was not entirely clear to me from Figure 1. Perhaps mapping the node numbers directly to the node, rather than a connected branch, would help? (I am assuming, based on nodes 1 and 2, that the labels are being placed on the branch directly antecedent to the node of interest)?

      Thank you so much for your suggestion. As pointed out, the calibrations used were applied at the crown node of existing Cyanobacterial clades, not at the stem of photosynthetic Cyanobacteria. We agree that photosynthesis and therefore the production of molecular oxygen may have been present in more ancient Cyanobacterial clades, however; these groups have not been discovered yet or went extinct. We have improved Fig. 1 to avoid confusion and now it is part of the updated version of our manuscript.

      Alleon J, Summons RE. 2019. Organic geochemical approaches to understanding early life. Free Radic Biol Med 140:103–112.

      Buick R, Dunlop JSR. 1990. Evaporitic sediments of Early Archaean age from the Warrawoona Group, North Pole, Western Australia. Sedimentology 37: 247-277.

      Creevey CJ, Doerks T, Fitzpatrick DA, Raes J, Bork P. 2011. Universally distributed single-copy genes indicate a constant rate of horizontal transfer. PLoS One 6:e22099.

      Djokic T, Van Kranendonk MJ, Campbell KA, Walter MR, Ward CR. 2017. Earliest signs of life on land preserved in ca. 3.5 Ga hot spring deposits. Nat Commun 8:15263.

      Giovannoni SJ, Stingl U. 2005. Molecular diversity and ecology of microbial plankton. Nature 437: 343-348. Hickman-Lewis K, Cavalazzi B, Giannoukos K, D'Amico L, Vrbaski S, Saccomano G, et al. 2023. Advanced two-and three-dimensional insights into Earth's oldest stromatolites (ca. 3.5 Ga): Prospects for the search for life on Mars. Geology 51: 33-38.

      Lollar BS, McCollom TM. 2006. Geochemistry: biosignatures and abiotic constraints on early life. Nature. Martinez-Gutierrez CA, Aylward FO. 2021. Phylogenetic Signal, Congruence, and Uncertainty across Bacteria and Archaea. Mol Biol Evol 38:5514–5527.

      Ren M, Feng X, Huang Y, Wang H, Hu Z, Clingenpeel S, Swan BK, Fonseca MM, Posada D, Stepanauskas R, Hollibaugh JT, Foster PG, Woyke T, Luo H. 2019. Phylogenomics suggests oxygen availability as a driving force in Thaumarchaeota evolution. ISME J 13:2150–2161.

      Walter M R, R Buick, JSR Dunlop. 1980. Stromatolites 3,400–3,500 Myr old from the North pole area, Western Australia. Nature 284: 443-445.

      Wu M, Scott AJ. 2012. Phylogenomic analysis of bacterial and archaeal sequences with AMPHORA2. Bioinformatics 28:1033–1034.

      Yang Y, Zhang C, Lenton TM, Yan X, Zhu M, Zhou M, Tao J, Phelps TJ, Cao Z. 2021. The Evolution Pathway of Ammonia-Oxidizing Archaea Shaped by Major Geological Events. Mol Biol Evol 38:3637–3648.

  6. Oct 2023
    1. Author Response

      The following is the authors’ response to the original reviews.

      We thank the editor and the reviewers for their very useful and constructive comments. We went through the list and gladly received all their suggestions. The reviewers mostly pointed to minor revisions in the text, and we acted on all of those. The one suggestion that required major work was the one raised in point 13, about the processing pipeline being unconvincingly scattered between different tools (R → Python → Matlab). I agree that this was a major annoyance, and I am happy to say we have solved it integrating everything in a recent version of the ethoscopy software (available on biorxiv with DOI https://www.biorxiv.org/content/10.1101/2022.11.28.517675v2 and in press with Bioinformatics Advances). End users will now be able to perform coccinella analysis using ethoscopy only, thus relying on nothing else but Python as their data analysis tool. This revised version of the manuscript now includes two Jupyter Notebooks as supplementary material with a “pre-cooked” sample recipe of how to do that. This should really simplify adoption and provides more details on the pipeline used for phenotyping.

      Please find below a point-by-point description of how we incorporated all the reviewers’ excellent suggestions.

      Recommendations for the authors: please note that you control which, if any, revisions, to undertake

      1) Line 38: "collecting data simultaneously from a large number of individuals with no or limited human intervention" is a bit misleading, as the entire condition the individuals are put in are highly modified by humans and most times "unnatural". I understand the point that once the animals are placed in these environments, then recording takes place without intervention, but it would be nice to rephrase this so that it reflects more accurately what is happening.

      We have now rephrased this into the following (L39):

      Collecting data simultaneously from a large number of individuals, which can remain undisturbed throughout recording.

      2) Line 63: please add a reference to the Ethoscopes so that readers can easily find it.

      Done.

      2b) And also add how much they cost and the time needed to build them, as this will allow readers to better compare the proposed system against other commercially available ones.

      This information is available on the ethoscope manual website (http://lab.gilest.ro/ethoscope). The price of one ethoscope, provided all necessary tools are available, is around ~£75 and the building time very much depends on the skillset of the builder and whether they are building their first ethoscope or subsequent ones. In our experience, building and adopting ethoscopes for the first time is not any more time-expensive than building a (e.g.) deeplabcut setup for the first time. We have added this information to L81

      Ethoscopes are open source and can be manufactured by a skilled end-user at a cost of about £75 per machine, mostly building on two off-the-shelf component: a Raspberry Pi microcomputer and a Raspberry Pi NoIR camera overlooking a bespoke 3D printed arena hosting freely moving flies.

      3) Line 88: The authors describe that in the current setting, their system is capable of an acquisition rate of 2.2 frames per second (FPS). Would reducing the resolution of the PiCamera allow for higher FPS? I raise this point because the authors state that max velocity over a ten second window is a good feature for classifying behaviors. However, if animals move much faster than the current acquisition rate, they could, for instance, be in position X, move about and be close to the initial position when the next data point is acquired, leading to a measured low max velocity, when in fact the opposite happened. I think it would be good to add a statement addressing this (either data from the literature showing that the low FPS does not compromise data acquisition, or a test where increasing greatly FPS leads to the same results).

      We have previously performed a comparison of data analysed using videos captured at different FPSs, which is published in Quentin Geissman’s doctoral Thesis (2018, DOI: https://doi.org/10.25560/69514 ) in chapter 2, section 2.8.3, figure 2.9 ). We have now added this work as one of the references at L95 (reference 19).

      4) Still on the low FPS, would a Raspberry Pi 4 help with the sampling rate? Given that they are more powerful than the RPi3 used in the paper?

      It would, but it would be a minor increase, leading from 2.2 to probably 3-5 FPS. A significantly higher number of FPSs would be best achieved by lowering the camera’s resolution, as the reviewer’s suggested, or by operating offline. I think the interesting point being implied by the reviewers is that, for Drosophila, the current limits of resolution are more than sufficient. For other animals, perhaps moving more abruptly, they may not. The reviewer is right that we should add a line of caveat about this. We now do so in the discussion, lines 215-224.

      Coccinella is a reductionist tool, not meant to replace the behavioural categorization that other tools can offer but to complement it. It relies on raspberry PIs as main acquisition devices, with associated advantages and limitations. Ethoscopes are inexpensive and versatile but have limitations in terms of computing power and acquisition rates. Their online acquisition speed is fast enough to successfully capture the motor activity of different species of Drosophilae28, but may not be sufficient for other animals moving more swiftly, such as zebrafish larvae. Moreover, coccinella cannot apply labels to behaviour (“courting”, “lounging”, “sipping”, “jumping” etc.) but it can successfully identify large behavioural phenotypes and generate unbiased hypothesis on how behaviour – and a nervous system at large – can be influenced by chemicals, genetics, artificial manipulations in general.

      5) Along the same line of thought, would using a simple webcam (with similar specs to the PiCamera - ELP has cameras that operate on infrared and are quite affordable too) connected to a more powerful computer lead to higher FPS? - The reason for the question about using a simple webcam is that this would make your system more flexible (especially useful in the current shortage of RPi boards on the market) lowering the barrier for others to use it, increasing the chances for adoption.

      Completely bypassing ethoscopes would require the users to setup their own tracking solution, with a final result that may or may not match what we describe here. If a greater temporal resolution is necessary, the easiest way to achieve more FPSs would be to either decrease camera resolution or use the Pis to take videos offline and then process those videos at a later stage. The combination of these two would give FPS acquisition of 60 fps at 720p, which is the maximum the camera can achieve. We now made this clear at lines 83-92.

      The temporal and spatial resolution of the collected images depends on the working modality the user chooses. When operating in offline mode, ethoscopes are capable to acquire 720p videos at 60 fps, which is a convenient option with fast moving animals. In this study, we instead opted for the default ethoscope working settings, providing online tracking and realtime parametric extraction, meaning that images are analysed by each raspberry Pi at the very moment they were acquired (Figure 1b). This latter modality limits the temporal resolution of information being processed (one frame every 444 ms ± 127 ms, equivalent to 2.2 fps on a Raspberry Pi3 at a resolution of 1280x960 pixels with each animal being constricted in an ellipse measuring 25.8 ± 1.4 x 9.85 ±1.4 pixels - Figure 1a) but provides the most affordable and high-throughput solution, dispensing the researcher from organising video storage or asynchronous video processing for animals tracking.

      6) One last point about decreasing use barrier and increasing adoption: Would it be possible to use DeepLabCut (DLC) to simply annotate each animal (instead of each body part) and feed the extracted data into your current analysis with coccinella? This way different labs that already have pipelines in place that use DLC would have a much easier time in testing and eventually switching to coccinella? I understand that extracting simple maximal velocity this way would be an overkill, but the trade-off would again be a lowering of the adoption barrier.

      It would certainly be possible to calculate velocity from the whole animal pose measurement and then use this with HCTSA or Catch22, thus mimicking the coccinella pipeline, but it would be definitely overkilled, as the reviewers correctly points out. Given that we are trying to make an argument about high-throughput data acquisition I would rather not suggest this option in the manuscript.

      7) Line 96: The authors state that once data is collected, it is put through a computational frameworkthat uses 7700 tests described in the literature so that meaningful discriminative features are found. I think it would be interesting to expand a bit on the explanation of how this framework deals multiple comparison/multiple testing issues.

      We always use the full set of features on aggregate to train a classifier (e.g., TS_Classify in HCTSA) and that means no correction is necessary because the trained classifier only ever makes a single prediction (only one test is performed), so as long as it is done correctly (e.g., proper separation of training and test sets, etc.) then multiple hypothesis correction is not appropriate. This has been confirmed with the HCTSA/Catch22 author (Dr Ben Fulcher, personal communication). We have added a clarifying sentence about this to the methods (L315-318)

      8) It would be nice to have a couple of lines explaining the choice of compounds used for testing and also why in some tests, 17 compounds were used, while in others 40, and then 12? I understand how much work it must be in terms of experiment preparation and data collection for these many flies and compounds, but these changes in the compounds used for testing without a more detailed explanation is suboptimal.

      This is another good point. We have now added this information to the methods, in a section renamed “choice, handling and preparation of drugs” L280-285, which now reads like this:

      The initial preliminary analysis was conducted using a group of 12 compounds “proof of principle” compounds and a solvent control. These compounds were initially used to compare both the video method and ethoscope method. After testing these initial compounds, it was found that the ethoscope methodology was more successful, and then the compound list was expanded to 17 (including the control) only using the ethoscope method. As a final test, we included additional compounds for a single concentration, bringing up the total to 40 (including control), also for the ethoscope method.

      9) Line 119 states: "A similar drop in accuracy was observed using a smaller panel of 12 treatments (Supplementary Figure 2a)". It is actually Supplementary Figure 1c.

      Thank you for noticing that! Now corrected. The Supplementary figures have also been renamed to obey eLife’s expected nomenclature (both Figure 1 – Figure supplements)

      10) In some places the language seems a little outlandish and should either be removed or appropriately qualified. a- Lines 56-59 pose three questions that are either rhetorical or ill-posed. For example, "...minimal amount of information...behavior" implies there is a singular response but the response depends on many details such as to what degree do the authors want to "classify behavior".

      Yes, those were meant as rhetorical questions indeed, but we prefer to keep them in, because we are hoping to generate this type of thoughts with the readers. These are concepts that may not be so obvious to someone who is just looking to apply an existing tool and may spring some reflection about what kind of data do they really want/need to acquire.

      b) Some of the criticisms leveled at the state-of-the-art methods are probably unwarranted because the goals of the different approaches are different. The current method does not yield the type of rich information that DeepLabCut yields. So, depending on the application DeepLabCut may be the method of choice. The authors of the current manuscript should more clearly state that.

      In the introduction and discussion we do try to stress that coccinella is not meant to replace tools like DLC. We have now added more emphasis to this concept, for instance to L212:

      [tools like deeplabcut] are ideal – and irreplaceable – to identify behavioural patterns and study fine motor control but may be undue for many other uses.

      And L215:

      Coccinella is a reductionist tool not meant to replace the behavioural categorization that other tools can offer but to complement it

      11) The application to sleep data appears suddenly in the manuscript. The authors should attempt to make with text change a smoother transition from drug screen to investigation into sleep.

      I agree with this observation. We have now tried to add a couple of sentences to contextualise this experiment and hopefully make the connection appear more natural. Ultimately, this is a proof-ofprinciple example anyway so hopefully the reader will take it for what it is (L169).

      Finally, to push the system to its limit, we asked coccinella to find qualitative differences not in pharmacologically induced changes in activity, but in a type of spontaneous behaviour mostly characterised by lack of movement: sleep. In particular, we wondered whether coccinella could provide biological insights comparing conditions of sleep rebound observed after different regimes of sleep deprivation. Drosophila melanogaster is known to show a strong, conserved homeostatic regulation of sleep that forces flies to recover at least in part lost sleep, for instance after a night of forceful sleep deprivation.

      11b) Additionally, the beginning section of sleep experiments talks about sleep depth yet the conclusion drawn from sleep rebound says more about the validity of the current 5 min definition of sleep than about sleep depth. If this conclusion was misunderstood, it should be clarified. If it was not, the beginning text of the sleep section should be tailored to better fit the conclusion.

      I am afraid we did not a good job at explaining a critical aspect here: the data fed to coccinella are the “raw” activity data, in which we are not making any assumption on the state of the animal. In other words, we do not use the 5-minutes at this or any other point to classify sleep and wakening. Nevertheless, coccinella picks the 300 seconds threshold as the critical one for discerning the two groups. This is interesting because it provides a full agnostic confirmation of the five minutes rule in D. melanogaster. We recognise this was not necessarily obvious from the text and now added a clarification at L189-201:

      However, analysis of those same animals during rebound after sleep deprivation showed a clear clustering, segregating the samples in two subsets with separation around the 300 seconds inactivity trigger (Figure 3d). This result is important for two reasons: on one hand, it provides, for the third time, strong evidence that the system is not simply overfitting data of nought biological significance, given that it could not perform any better than a random classifier on the baseline control. On the other hand, coccinella could find biologically relevant differences on rebound data after different regimes of sleep deprivation. Interestingly enough, the 300 seconds threshold that coccinella independently identified has a deep intrinsic significance for the field, for it is considered to be the threshold beyond which flies lose arousal response to external stimuli, defining a “sleep quantum” (i.e.: the minimum amount of time required for transforming inactivity bouts into sleep bouts23,24,28). Coccinella’s analysis ran agnostic of the arbitrary 5-minutes threshold and yet identified the same value as the one able to segregate the two clusters, thus providing an independent confirmation of the fiveminutes rule in D. melanogaster.

      12) Line 227: (standard food) - please add a link to a protocol or a detailed description on what is "standard food". This way others can precisely replicate what you are using. This is not my field, but I have the impression that food content/composition for these animals makes big changes in behaviour?

      Yes, good point. We have now added the actual recipe to the methods L240:

      Fly lines were maintained on a 12-hour light: 12-hour dark (LD) cycle and raised on polenta and yeast-based fly media (agar 96 g, polenta 240 g, fructose 960 g and Brewer’s yeast 1,200 g in 12 litres of water).

      13) Data acquisition and processing: please add links to the code used.

      Both the code and the raw data used to generate all the figures have been uploaded on Zenodo and available through their repository. Zenodo has a limit of 50GB per uploaded dataset so we had to split everything into two files, with two DOIs, given in the methods (L356, section “code and availability” - DOIs: 10.5281/zenodo.7335575 and 10.5281/zenodo.7393689). We have now also created a landing page for the entire project at http://lab.gilest.ro/coccinella and linked that landing page in the introduction (L64).

      13b) Also your pipeline seems to use three different programming languages/environments... Any chance this could be reduced? Maybe there are R packages that can convert csv to matlab compatible formats, so you can avoid the Python step? (nothing against using the current pipeline per se, I am just thinking that for usability and adoption by other labs, the smaller amount of languages, the better?

      This is a very important suggestion that highlights a clear limitation of the pipeline. I am happy to say that we worked on this and solved the problem integrating the Python version of Catch22 into the ethoscopy software. This means the two now integrate, and the entire analysis can be run within the Python ecosystem. HCTSA does not have a Python package unfortunately but we still streamlined the process so that one only has to go from Python to Matlab without passing through R. To be honest, Catch22 is the evolution of HCTSA and performs really well so I think that is what most users will want to use. We provide two supplementary notebooks to guide the reader through the process. One explains how to go from ethoscope data to an HCTSA compatible mat file. The other explains how ethoscope data integrate with Catch22 and provides many more examples than the ones found in the paper figures.

      14) There are two sections named "References" (which are different from each other) on the manuscript I received and also on BioRxiv. Should one of them be a supplementary reference? Please correct it. I spent a bit of time trying to figure out why cited references in the paper had nothing to do with what was being described...

      The second list of references actually applied only to the list of compounds in the supplementary table 1. When generating a collated PDF this appeared at the end of the document and created confusion. We have now amended the heading of that list in the following way, to read more appropriately:

  7. Sep 2022
    1. Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      The authors have assembled an enormous amount of statistical data on the genomes and phylogeny of Arctic algae, including the genomes of four new species that they sequenced for this study. Their main finding is that horizontal gene transfer has led to convergent evolution in distantly related microalgae.

      **Major comments**

      Reviewer #1__: The purpose of the study is not clearly stated in the abstract or the introduction. The authors say (line 93) "Defining the genetic adaptations underpinning these small algal species is crucial as a baseline to understand their response to anthropogenic global change (Notz & Stroeve,2016)." Is this their goal? Or are they just quoting another study? The authors state (line 103) "We extend by sequencing the genomes of four distantly related microalgae...". This is not really a question or a hypothesis. I am sure the authors can provide a more compelling reason to embark on such a labor-intensive study.__

      Reply: We agree that the aim was lost in the details and the Introduction is now focused towards the original goal of the study, which was to investigate convergent evolution in a biogeographically isolated ocean. Additional references on the formation and history of the Arctic Basin have been added to the Introduction to provide context. “An ocean has been present at the pole since the beginning of the Cretaceous. Shaped by tectonic processes (Nikishin et al., 2021) the Arctic Ocean has been a relatively closed basin since the Masstrichtian at the end of the late Cretaceous epoch (ca. 70 million years before present), with episodic sea-ice cover since that time (Niezgodzki et al., 2019). This long history suggests limited gene flow from the global ocean over vast time scales and Arctic marine species including microalgae could well have unique adaptations to cold arctic conditions.” Line 78-83.

      And following this we provide a clear hypothesis “The potential for lineages of ancient Arctic origin and the episodic input of outside species led us to our hypothesis that Arctic microalgae convergently evolved traits or adaptations aiding survival in an ice-influenced ocean. Line 112-117.

      We also discuss both the adaptive and distinct physical environment of the Arctic, and its topographical separation from other ocean regions as dispersal limitation would enhance the Arctic-specific genomic signatures. We now cite the recent paper by Sommeria-Kline et al. (2020), which puts eukaryotic plankton biogeography into a global context (Line 72)

      Reveiwer #1__: The most prominent shared trait that the authors found are genes for ice-binding proteins. However, in view of their importance, little information is given about their different types and possible functions.__

      Reply: We appreciate the comment and have added information on relevant ice binding proteins found in the Arctic Algae. In addition, we discuss how the functional and secretory diversity of IBP would enhance the survivability of pelagic taxa. Lines 534 to 564.

      Although ice binding proteins from multicellular animals and plants are outside the scope of this study, there is a recent review; Bar Doley, Braslavsky and Davies 2016 Annual review of Biochemisty 85: 515-542.

      .

      Reviewer #1__: The HGT of ice-binding proteins is a major focus of this study, but little is said about what previous studies have said about this. What are the previous studies, what are their findings and how do the present findings contribute to this?__

      Reply: We agree that this aspect should have been more visible. We incorporated new data to characterize IBPs drawn from MMETSP transcriptomes, and environmental Tara Ocean metagenomes, as well as our Arctic strains. We note that as we take a PFAM-based approach, the IBPs treated are DUF3494/PF11999 domain, which are type 1 IBPs / algal IBPs (Raymond and Remia 2019). As an example of novelty, we identify the position of IBPs from dinoflagellates, within a larger Arctic Clade that included CCMP2293, CCMP2436 and CCMP2097 and Arctic TARA IBP, rendering this a pan-algal IBD clade.

      In addition, we were able to resolve the position of anomalous F. cylindrus IBP that fell between two Arctic associated clades (A and B, in our Fig 4). This finding is consistent with F. cylindrus originating in the Arctic as previously suggested and subsequently invading the Southern Ocean.

      The recurrent acquisition of multiple diverse IBP isoforms in individual species through HGT events has not been previously reported, and the extent of isoforms in the Arctic was surprising. See for example multiple different IBP forms with separate origins in Pavlovales CCMP2436 (Fig 4). The previous studies are referred to in the context of the phylogeny of the IBD within the results section: Lines 322- 413, and Lines 534-585.

      Reviewer #1: Figure 5 on HGT of ice-binding proteins is difficult to follow. It would be clearer if each panel could be described separately, clearly stating its main finding. I doubt that a reader could look at this figure and explain to a colleague what it shows.

      Reply: We have revised rearranged the figure (now Fig 4) with Arctic A, B, C and D clearly indicated as well as the two Antarctic dominated clades. The upper schematic includes the deepest phylogeny of algal IBDs to date, incorporating all of UniRef, MMETSP and TARA Oceans. The fasta files underlying the tree and the nexus file used are provided the S1 Data Folder, which is an excel folder with information on the analysis of the data. The callout and order of the clades has been revised to facilitate interpretation of the phylogenies more clearly. The entire section has been completely rewritten.

      Reviewer #1: This is also a problem with many of the other figures. For each figure, what is the question being asked and what is its take-home message?

      Reply: We agree that the message was lost and have now focused on our original question in our accepted proposal to JGI. “Is there a convergence among arctic microalgae at the genomic level?”. We found some genome properties were common among the Arctic isolates (more unknown PFAMS and several expanded PFAMs). The importance of ice binding proteins in Arctic Isolates and the widespread inter-algal HGT of this important protein among the Arctic strains. The IBP biogeography and phylogeny strongly indicate that the Arctic microalga have acquired IBP locally and that the Antarctic strains have acquired additional isoforms independently from Antarctic bacteria and fungi (Lines 565-585).

      Reviewer ____#1____: ____The paper has more data than a reader can absorb. It could be strengthened by reducing the number of figures, simplifying them if possible, and more clearly stating the value of the remaining figures.

      Reply. As suggested, we have refocused the paper, removing more speculative statistics based analysis and associated figures. The main conclusions are supported by the 5 main figures. We are now present 5 main figures and 11 supplementary figures (previously 23 downloadable supplementary figures and 40 on-line only figures supporting the support figures). We agree with the reviewer, and we feel the revised version is a more transparent synthesis. Briefly the Figures illustrate the following points. Fig. 1. The multigene tree of available algal genomes and transcriptomes provides a clear framework for judging the divergence of subsequent individual gene and PFAMs phylogenies. Fig. 2 (originally Fig. 3). Indicates the convergence of PFAM domains in the Arctic strains, in contrast to strains from elsewhere. Fig. 3 (originally Figure 4) shows Arctic specific expansions and contraction of PFAM domains, again demonstrating convergent evolution in the Arctic. The figure identifies specific PFAMs that contribute to the within-Arctic convergence. This figure is based on statistical methods independent of Fig 2. Figure 4 is the most extensive IBP phylogeny to date and has been discussed above. Figure 5, which was supplementary in our non-peer reviewed version, shows the biogeographic distribution of IBP, and can be compared to the distributions of the 18S rRNA genes from the four Arctic algae provided as supplementary (S6 Fig.)

      **Minor comments**Reviewer #1

      1. The figure citations are confusing. E.g., what does "Fig.1- Figure supplement 1" refer to? Does this refer to 1 or 2 figures? Apparently, it refers only to Fig. S1, so many readers will be confused when they look at Fig. 1.

      Reply: We apologize for the confusing format; the manuscript had been formatted for the online journal eLife. Our revision follows the more traditional style of PLoS Biology and other Review Commons journals.

      .

      Multiple citations should be in order of publication date, not alphabetical order.

      Reply ; We agree that date of publications is quite standard and recognizes priority of publication. Several on line journals no longer follow this rule and citation order will follow the specific style used by our accepting journal.

      Reviewer #1 (Significance (Required)): It is well known that useful genes tend to be shared among microorganisms. The present study strengthens previous studies in showing that gene transfer is an important process in polar regions.

      Reply: We thank the reviewer for recognizing the importance of our study.


      Reviewer #2 ____(Evidence, reproducibility, and clarity (Required)):

      This manuscript is the result of a large international collaborative effort, including the US Department of Energy Joint Genome Institute. Its focus is comparative genomics of eukaryotic Arctic algae. The primary data described in the ms are four new genome and transcriptome sequences from diverse Arctic algae, represented by a cryptomonad, a haptophyte, a chrysophyte, and a pelagophyte.

      The authors compare these new data to previously published genomic/transcriptomic data from eukaryotic algae with the goal of understanding genome evolution in the Artic. The results of the paper are a series large-scale comparative genomic bioinformatics analyses, including the associated statistical analyses. The key findings center on statistically significant features of Arctic genomes, features that stand out as compared to the genomes of algae that are not primarily found in the Arctic. Together, these findings allow the authors to make various hypotheses and suggestions about genetic adaptations to polar environments.

      By far the most significant finding is that the genomes of Arctic algae are enriched in genes encoding proteins with an ice-binding domain, paralleling findings from Antarctic algae. These genes appear to have spread among Arctic algal genomes via horizontal gene transfer, which raises a series of interesting questions. In my opinion, the major conclusions of this paper are supported by the data. Listed below are a few comments that may improve the ms:

      Reviewer #2.

      1) In today's post-genomics era, everyone seems to be sequencing nuclear genomes. Often what distinguishes high-impact and low-impact genome papers is the number of genomes presented and the quality of the genome assembly. I may have missed it, but reading the main text, the figures/tables, and the supplementary data I was not able to get a sense of the quality of the four genome assemblies from which the main findings are based. I was eventually able to find this information from PhycoCosm (note: some of the links to this site are not working in the ms). My quick scan of the PhycoCosm summary info for the four genomes indicates that the assemblies are highly fragmented, likely because they are based on short-read Illumina sequencing rather than a combination of short and long reads. I think it is important to briefly discuss (and or present) the quality of the assemblies in the ms and to highlight the potential limitations/drawbacks of employing highly fragmented assemblies when carrying out large-scale comparative genomics.

      Reply: We agree and the data concerning the genome quality assemblies has been moved to the main text Table 1. The comparison with other paired related strains is provided in an excel folder designated S2 Data Folder.

      Reviewer #2.

      2) Horizontal gene transfer is undeniably a major driving force in evolution, and one that has shaped genomic architecture across the Tree of Life. I believe the data presented here support a role for HGT in the genome of evolution of Arctic algae, particularly with respect to genes encoding proteins with an ice-binding domain. However, we can all think of numerous instances when authors of genome papers were too quick to point to HGT. Thus, I would urge more caution and balance when presenting the HGT data, including some discussion about factors that could incorrectly lead researchers to conclude a significant role for HGT, such as contamination, gene duplication, mis-assemblies, etc. I'm not suggesting that you change the main conclusions, but just tone down the language in places (e.g., "we reveal remarkable convergence in the coding content ... ").

      Reply: We understand the reviewers concerns and now more clearly outline the pipeline we have used to identify HGTs. This included: filtering each genome to remove all possible contaminant sequences first, considering both contig co-presence of vertical- and horizontally-derived genes, and reciprocal and independent annotations of gene sequences in both genome sequences and MMETSP transcriptomes. Retained genes were subjected to simultaneous BLAST analysis and manually curated phylogenies using decontaminated reference datasets. The most parsimonious explanation for our final IBP domain microbial algal clusters (Fig 4) is HGT. On the side of caution, we removed the entire section that identified potential arctic HGT based primarily on a less targeted broad statistical analysis. The focus is now on 3 genes that have clearly identifiable utility in the Arctic, were found to be enriched in Arctic genomes via a separate analysis and had homologs in the Tara Ocean Polar circle data. In addition, we describe more clearly the role of expansion and enrichment of PFAMs and the high proportion genes without an identifiable PFAMs in the Arctic strains as evidence for Arctic convergence separate from potential HGT.

      Reviewer #2.

      3) The downside of studying protists (as compared to multicellular animals, for instance) is that most are not widely known by the scientific community and even fewer scientists can picture what they actually look like (e.g., Pavlovales sp. CCMP2436). A few more details about the four Arctic algae that make up the focus of this paper might be helpful for the casual reader. My sense is that if at the next departmental meeting I asked my colleagues what a pelagophyte was most would look at me with a blank stare. Moreover, am I right to assume that all four algae are psychrotolerant rather than psychrophilic (Supplement Fig. 1 makes me think otherwise). It might be good to point out the difference in the text.

      Reply: High resolution images of each strain are available on the JGI home page for each alga, given the multiple figures we feel photos would not add information.

      Reviewer #2

      4) I don't think Supp. Table 1 (the Pan-algal dataset) got uploaded correctly during the manuscript submission stage. The first link I click on gives me Supp. Table 2.

      Reply: We apologize for this, the format was incorrect for the file designation and there were lost links. We now more actually refer to these as Data Folders as they are excel folders containing multiple sheets, All supplementary links will be verified again on final submission.

      .

      Reviewer #2 (Significance (Required)):

      By far the most significant finding from this paper is that the genomes of Arctic algae are enriched in genes encoding proteins with an ice-binding domain, paralleling findings from Antarctic algae. These genes appear to have spread among Arctic algal genomes via horizontal gene transfer, which raises a series of interesting questions. This is not the first paper to present these types of ideas, but it is arguably the broadest analysis yet, at least with respect to eukaryotic algae. This work will be of great interest to polar scientists, phycologists, protistologists, and the genomics community. I am genome scientist studying protists, including algae.

      Reply. We thank the reviewer for their insightful comments.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      **Summary:**

      This manuscript is focused on Arctic microalgae, an important yet understudied community in permanently cold ecosystems. By sequencing the genomes of four phylogenetically diverse and uncharacterized polar algae, the authors seek to elucidate genomic features and protein families that are similar in polar species (and differ from their relatives from temperate environments) This work used high-throughput genomic sequencing and computational analysis to demonstrate significant horizontal gene transfer (HGT) in several gene families, including ice-binding proteins. The authors suggest that this HGT is an effector of environmental adaptation to Arctic environments.

      **Major comments and experiment suggestions:**

      The authors conclude that HGT between arctic species is a driver of polar adaptation. The authors strongly support the claim that HGT is present more frequently in the polar algae examined here. Whether this is adaptive should be further explored though. For instance, ice-binding domains were one PFAM group found at significantly higher frequencies in the polar species - but are all of these species associated with ice? What would be the benefit of IBDs in an alga that is found in the open ocean. Similar with the other domains (Lns 333-335), its not clear whether these are truly adaptive features. ____This is more speculative.

      Reply: We agree that detail was lacking and have considerably expanded our introduction on the character of the Arctic Ocean and have stated the goals and underlying hypothesis. Briefly, all surface water organisms that live in the Arctic encounter ice during the year as the ocean freezes in winter, and surface waters reman around negative 1.7 °C for much of the year. This information has been added to the introduction. We have also expanded the discussion on the multiple effects of different IBPs that would be ecologically beneficial for plankton as well as ice-algae and cite relevant experimental studies and reviews.

      Reviewer #3) ____HGT was a major conclusion of this study, putting this in a wider perspective would strengthen the conclusion, especially in the context of HGT from prokaryotes. Are there insights on whether IBDs are present in Arctic prokaryotes?

      Reply: This is a good question, and we now point out that there were 91 Arctic bacterial and archaeal IBP sequences in our comparative dataset. In contrast to the Antarctic clades, none were closely related to the Arctic strain IBPs (Fig 4). Line 336.

      Reviewer #3) ____The data obtained from the genomic works supports the conclusions stronger that ones from transcriptomes, where what genes/domains are present would depend largely on the sampling conditions. This should be emphasized.

      Reply: The main rational for using transcriptomes was that more of these are available and enabled us to detect convergences and HGT across a broader taxonomic range than would be possible with genome-only data, where we had access to a total of only 21 microalgal genomes. In general transcriptome studies are aimed at identifying responses under different conditions and rely on comparative expression data, usually 2-fold differences in up or down expression under different growth conditions, see for example Freyria et al. 2022 (Communications Biology). Unlike a transcriptome expression study, our data mining detected any (constitutive or regulated) expression in these unicellular haploid cells, we would have detected genes used under any condition that an algal happened to be growing. IBD was not detected in any of the temperate genomes, and only detected in transcriptomes of Arctic and Arctic-Boreal groups. However, we agree that there may be some limitation of transcriptomes only studies and mention this. Lines 522-528.

      Reviewer #3) ____An experiment to determine whether the species are cold extremophiles (psychrophiles) would be useful here to strongly support the data in Figure 1. The authors state that their species can not survive >6C but this is based on experiments done on older studies. Considering the cultures have been maintained as a continuous culture for decades, confirming that they still have psychrophilic characteristic would be useful. This is a straightforward and low cost experiment that requires simply measuring growth rates at several temperatures to define the optimal and confirm that the cells are not viable above 6C.

      Reply: These are interesting points, and the broad “background” statements in the original manuscript would require a separate study,and have been deleted. Temperature tolerance experiments are not so simple for cold adapted algae with slow growth rates. Such experiments require specialized incubators to maintain low temperatures. Temperature experiments have been carried out on the cultures in the context of other studies, see for example, Daugberg et al. 2018, J. Phycol. But this is not within the scope of the present study.

      We now restrict our conclusions to the specific question of convergence among Arctic strains. We apologize for the misunderstanding on the history of the cultures. They have not been in “continuous culture” but are cryopreserved. We now simply indicate that they grow below 6 °C, which is sufficient to assume that they are likely cryophiles, our experience is that they do not grow well or at all at higher temperatures, our efforts have been to maintain the cultures that are otherwise easily lost. We now make no claims about optimality or limits. Here we simply examined genomes and available transcriptomes that were generated from algae growing at 4-6 °C.

      Reviewer #3) ____**Minor comments:**

      Defining the species used here as psychrophiles would put the study in context better. The authors relate their finding to Antarctic species (HGT, ice-binding domains, large genomes) all of which are confirmed psychrophiles.

      Reply: The temperature definition of psychrophiles is surprisingly high (optimal growth below 15 °C) and this definition of psychrophiles is now given in the introduction. The point is really that there are few isolates from cold surface waters that have been well studied. We now add. “A handful of polar algal genomes have been extensively studied, with 4 of these from around Antarctica and classified as psychrophiles (not being able to grow above 15 °C (Feller & Gerday, 2003)”. Lines 103-107.

      Reviewer #3) ____A short rationale on why these species at all would be useful - are they representative of their classes? Do they have psychrophilic characteristics that might make them useful models in the future? Are they widely used now?

      Reply: We appreciate the point as the definition of utility in discovery-based science is an open dialog.

      We agree that the study requires context and have added our rational for selecting the species for genome sequencing to the introduction. “To address questions on genetic adaptations to this ice-influenced environment, we sequenced 4 phylogenetically divergent microalgae, from 4 algal classes belonging to 3 algal phyla: Cryptophyceae (Cryptophyta), Pavlovophyceae (Haptophyta), Chrysophyceae and Pelagophyceae (both in the Ochrophyta) isolated from the ca. 77 °N, where surface ice flow persists through June (Mei et al., 2002). The four isolates were selected as representatives of different water and ice conditions and phylogeny from available strains collected in April and June 1998 during the North Water Polynya study”.

      Reviewer #3) ____Starting algal cultures were maintained in a continuous culture since 1998 and under continuous light since at least 2015, have the authors confirmed that these algae retain their physiological features even after this long time? The accumulation of mutations is a possibility here.

      Reply: We apologize for the misunderstanding of the timeline; the history of the cultures was not given in the manuscript and the inferred history is not quite correct. The 2015 date was the year of publication for the MMETSP data. Our continuous light statement is a record of our standard culture conditions. We now elaborate on the material used in the current study. The cultures were deposited in the Bigelow culture collection (now NCMA) in 2002 and cryopreserved once they had been verified and given a culture designation. We obtained fresh cultures in 2005 and these were used for the MMETSP project. We obtained fresh cultures again in 2011, specifically for the JGI genome project. These algae do not grow fast and most of the DNA was sent to JGI in 2012 for most of the isolates. This history is rather long and not relevant, since one would speculate that over the years the algae would tend to lose the ice associated functionality, e.g. they were not frozen in seawater every year for 4 to 6 months or subject to sudden freshwater exposure, when ice melts. We would encourage other researchers to order the cultures and run experiments. We note that many of the 40 or so algae isolated from the same campaign have been used by others for specific studies and at least 8 are in the MMETSP data set. The presence of 18S rRNA and phylogenetic position of the IBP sequences compared to Tara Arctic circle data confirms long-term Arctic presence of each species and the IBP domains in the Arctic without marked changes over the last 20 years.

      Reviewer #3) ____Ln381 - The culture collection IDs for each sequenced species should be included here

      Reply: we have added the culture IDs throughout.

      Reviewer #3) ____Ln. 389 - Algal cells are harvested and used for nucleic acid extraction, the nucleic acids themselves are not harvested

      Reply: we agree and corrected the wording

      Reviewer #3 (Significance (Required)):

      This study is well places in the current state of research on polar alga and represents a significant and very valuable addition to the current knowledge pool. Algae in general are lagging behind other groups of photosynthetic organisms in the number of sequenced and analyzed genomes, despite algae being one of the main primary producers globally. This is even more strongly felt in polar research, where only 4 species have been sequenced, most of which are restricted to Antarctica. There is a true gap in our knowledge when it comes to Arctic species, and this study fills this gap. As the authors correctly state, we need more knowledge on polar environments and the primary producers that support these important ecosystems in light of current climate change trends.

      Reply: we appreciate the succinct summary of our study and thank the reviewer for insights and suggestions that have improved the manuscript.

      Reviewer field of expertise: Polar algae, stress responses, plant and algal energetics, cell signalling

      Reply: We appreciate the incites and perspective steming from the reviewer's expertise.

      Relevant key references cited in the reply:

      Daugbjerg N, Norlin A, Lovejoy C. Baffinella frigidus gen. et sp. nov. (Baffinellaceae fam. nov., Cryptophyceae) from Baffin Bay: Morphology, pigment profile, phylogeny, and growth rate response to three abiotic factors. Journal of Phycology. 2018;54(5):665-80

      Feller, G. and Gerday, C. (2003) Psychrophilic enzymes: Hot topics in cold adaptation. Nat Rev Microbiol, 1, 200-208.

      Freyria NJ, Kuo A, Chovatia M, Johnson J, Lipzen A, Barry KW, et al. Salinity tolerance mechanisms of an Arctic Pelagophyte using comparative transcriptomic and gene expression analysis. Communications Biology. 2022;5(1). doi: 10.1038/s42003-022-03461-2

      Mei, Z. P., Legendre, L., Gratton, Y., Tremblay, J. E., Leblanc, B., Mundy, C. J., Klein, B., Gosselin, M., Larouche, P., Papakyriakou, T. N., Lovejoy, C. and Von Quillfeldt, C. H. (2002) Physical control of spring-summer phytoplankton dynamics in the North Water, April-July 1998. Deep-Sea Research Part Ii-Topical Studies in Oceanography, 49, 4959-4982.

      Niezgodzki, I., Tyszka, J., Knorr, G. and Lohmann, G. (2019) Was the Arctic Ocean ice free during the latest Cretaceous? The role of CO2 and gateway configurations. Global and Planetary Change, 177, 201-212.

      Nikishin, A. M., Petrov, E. I., Cloetingh, S., Freiman, S. I., Malyshev, N. A., Morozov, A. F., Posamentier, H. W., Verzhbitsky, V. E., Zhukov, N. N. and Startseva, K. (2021) Arctic Ocean Mega Project: Paper 3-Mesozoic to Cenozoic geological evolution. Earth-Science Reviews, 217.

  8. Jul 2022
    1. This also made me think of church bulletin ads, which all look the exact same way, except maybe it’s just a Catholic thing2?

      I thought of the same aesthetic as well, in part because it wasn't as "busy" as the comic book page aesthetic.

    1. https://www.zylstra.org/blog/2022/06/spring-83/

      I've been thinking about this sort of thing off and on myself.

      I too almost immediately thought of Fraidyc.at and its nudge at shifting the importance of content based on time and recency. I'd love to have a social reader with additional affordances for both this time shifting and Ton's idea of reading based on social distance.

      I'm struck by the seemingly related idea of @peterhagen's LindyLearn platform and annotations: https://annotations.lindylearn.io/new/ which focuses on taking some of the longer term interesting ideas as the basis for browsing and chewing on. Though even here, one needs some of the odd, the cutting edge, and the avant garde in their balanced internet diet. Would Spring '83 provide some of this?

      I'm also struck by some similarities this has with the idea of Derek Siver's /now page movement. I see some updating regularly while others have let it slip by the wayside. Still the "board" of users exists, though one must click through a sea of mostly smiling and welcoming faces to get to it the individual pieces of content. (The smiling faces are more inviting and personal than the cacophony of yelling and chaos I see in models for Spring '83.) This reminds me of Stanley Meyers' frequent assertion that he attempted to design a certain "sense of quiet" into the early television show Dragnet to balance the seeming loudness of the everyday as well as the noise of other contemporaneous television programming.

      The form reminds me a bit of the signature pages of one's high school year book. But here, instead of the goal being timeless scribbles, one has the opportunity to change the message over time. Does the potential commercialization of the form (you know it will happen in a VC world crazed with surveillance capitalism) follow the same trajectory of the old college paper facebook? Next up, Yearbook.com!

      Beyond the thing as a standard, I wondered what the actual form of Spring '83 adds to a broader conversation? What does it add to the diversity of voices that we don't already see in other spaces. How might it be abused? Would people come back to it regularly? What might be its emergent properties?

      It definitely seems quirky and fun in and old school web sort of way, but it also stresses me out looking at the zany busyness of some of the examples of magazine stands. The general form reminds me of the bargain bins at book stores which have the promise of finding valuable hidden gems and at an excellent price, but often the ideas and quality of what I find usually isn't worth the discounted price and the return on investment is rarely worth the effort. How might this get beyond these forms?

      It also brings up the idea of what other online forms we may have had with this same sort of raw experimentation? How might the internet have looked if there had been a bigger rise of the wiki before that of the blog? What would the world be like if Webmention had existed before social media rose to prominence? Did we somehow miss some interesting digital animals because the web rose so quickly to prominence without more early experimentation before its "Cambrian explosion"?

      I've been thinking about distilled note taking forms recently and what a network of atomic ideas on index cards look like and what emerges from them. What if the standard were digital index cards that linked and cross linked to each other, particularly in a world without adherence to time based orders and streams? What does a new story look like if I can pull out a card either at random or based on a single topic and only see it or perhaps some short linked chain of ideas (mine or others) which come along with it? Does the choice of a random "Markov monkey" change my thinking or perspective? What comes out of this jar of Pandora? Is it just a new form of cadavre exquis?

      This standard has been out for a bit and presumably folks are experimenting with it. What do the early results look like? How are they using it? Do they like it? Does it need more scale? What do small changes make to the overall form?


      For more on these related ideas, see: https://hypothes.is/search?q=tag%3A%22spring+%2783%22

  9. Jun 2022
    1. Pretty basic! And yet, noth­ing on the inter­net presently allows me to do this

      Pretty basic and pretty much what Jaiku did. It gave a somewhat holistic social platform view of people with the person in focus. You could see all the social platforms they shared on and what was shared, but also allowed you to turn off listening to platforms for that person.

      On the Jaiku webview I had a my own layer of how I had someone tagged (say foodie or Apple nerd) and I could click on that and seem others I followed who I believed had those interests. Which was a personal version of what I wrote up later as a Granular Social Network (https://www.vanderwal.net/random/entrysel.php?blog=1975).

    2. Ten mil­lion boards gives us a max­i­mum disk space require­ment of 22.17 gigabytes, eas­ily stored on a com­mod­ity hard drive or a cheap-enough cloud volume. A capa­ble com­puter could even hold that in RAM. Turns out, when you don’t store every user’s entire history, plus a record of every adver­tise­ment they’ve ever seen, your data­base can stay pretty slim!

      the limit on history is interesting and feels like a missed opportunity. in a world where storage is so cheap, we should be able to keep our own histories! thinking about sousveillance vs. surveillance and personal panopticons

    3. You might update your board twice an hour or twice a month; you might amend one sen­tence or reboot the whole thing. Pub­lish­ing a new ver­sion is instantaneous, as easy as tap­ping a button. You don’t have to man­age a server to pub­lish a board; you don’t even have to estab­lish an account on a server.

      reminds me of locket but web-based and with full user agency to create what they want. their own little pocket of the internet. Love the no account part and think that's super important..

    4. It was the expe­ri­ence of draft­ing the spec that changed my view, and my pace. Writing! Gets you every time!
    5. I will just observe that there is some­thing about this tech­nol­ogy that has seemed, over the years, to scold rather than invite; enclose rather than expand; and strip away rather than layer upon.
    6. Ten mil­lion boards gives us a max­i­mum disk space require­ment of 22.17 gigabytes, eas­ily stored on a com­mod­ity hard drive or a cheap-enough cloud volume. A capa­ble com­puter could even hold that in RAM. Turns out, when you don’t store every user’s entire history, plus a record of every adver­tise­ment they’ve ever seen, your data­base can stay pretty slim!

      This is essentially the SSB take too. I don't love it, but the way in which I don't love it is a crotchety computer professional way, not a meaningful one.

    7. Boards are cryp­to­graphically signed in such a way that they can be passed from server to server and, no mat­ter where your client gets a copy of a pub­lisher’s board, you can be assured it is valid.

      Peer to peer content transmission is an awfully big can of worms. Freedom to delete is real contentious in SSB/Fediverse sstuff.

    8. More importantly, every board holds its place, regard­less of when it was last updated.

      One uncomfortable thing about this is that it replicates a scarcity logic of space in an unscarce medium. I keep lots of barely-more-than-defunct feeds in my RSS reader because it costs me nothing to do so and I'd be so happy if they did start publishing again. Looking at an unchanged board for a year would feel different.

    9. I recommend this kind of project, this fla­vor of puzzle, to any­one who feels tan­gled up by the present state of the inter­net. Pro­to­col design is a form of inves­ti­ga­tion and critique. Even if what I describe below goes nowhere, I’ll be very glad to have done this think­ing and writing. I found it chal­leng­ing and energizing.

      This is fun! Maybe I should try sketching some stuff out here before I work further on that web directory SSG template project.

    10. Client appli­ca­tions dis­play all the boards you are fol­lowing together, lay­ing them out on a 2D canvas, pro­duc­ing unplanned juxtapositions, just like the news­stand above.

      Reminds me of the neocities webgardens (that list of creators is incomplete), doing something like this with iframes and friendly convention.

    11. unable to exe­cute JavaScript or load exter­nal resources, but oth­er­wise unrestricted. Boards invite pub­lish­ers to use all the rich­ness of mod­ern HTML and CSS.

      But -- external images? No images? 😔

    12. You prob­a­bly reached this web page through an email.

      looks around shiftily at RSS reader

    13. For my part, I believe presentation is fused to content; I believe pre­sen­ta­tion is a form of con­tent; so RSS can­not be the end of the story.

      Viva! I do still use RSS as more of a notification stream than a consumption stream; I don't really want the consumption to all happen in one place. When Cinni updates in her RSS feed, it's links to her proper pages, and this is right and good because her site is beautiful in a way RSS couldn't (shouldn't) accommodate.

    14. when a user stops speaking, they disappear, and, by corollary, as a fol­lower, you mostly encounter the users who are speak­ing nonstop.

      Hmm. I don't know that there's a way to get around the 90-9-1 ratios there. I had to resort to user scripts even for my own stuff. I'm reminded of this whole thing, too.

    15. I am not shar­ing, at this time, code for a client or server, although I have ref­er­ence imple­mentations of both that I’m test­ing with a cou­ple of friends.

      I am forcibly reminded of this orange site comment that makeworld had shared though I guess I haven't really gotten it out of my head in the first place.

    16. 2217 bytes

      why that number?

    17. For my part, I believe presentation is fused to content; I believe pre­sen­ta­tion is a form of con­tent; so RSS can­not be the end of the story.

      This is probably where Fraidycat falls down. But still, as you don't mention it, Robin, are you familiar with it?

    18. fol­low peo­ple

      Just follow? Not interact with? Build relationships with?

    19. And yet, noth­ing on the inter­net presently allows me to do this

      First thought: https://fraidyc.at/

      But I expect the discussion below to clarify how that does not suffice (?).

    20. What do you want from the inter­net, anyway?

      Literally laughed out loud to this.

    21. It needs, instead, close con­sid­er­a­tion and gen­er­ous imagination.

      This seems like a good opportunity to try hypothes.is again. I've been meaning to get back to using it. As an experiment, I'm going to try and annotate this post as I read it.

    1. spring ’83 boards are so inherently creative, it’s beautiful to have that kind of customisation not only encouraged but really forced

      is it? I feel like you could certainly participate with raw markdown styled with Robin's defaults. hard to know how people who aren't Like Us would use the form because of course it's people Like Us who want to

    1. But the profound magic trick of the signature: that it allows a piece of content to flow around the internet, handed from peer to peer, impossible to tamper with... it's too good to pass up.

      I wonder if, given that boards are supposed to expire anyway, the key rotation shouldn't involve publishing old secrets such that it's not possible to use an old key to pin someone down as having published something (which would be long after its intended expiry anyway)? With the caveat that I don't know nothing about cryptography, I'm just a horrible little goblin typing in a Hypothes.is pane.

    2. New boards should be transmitted to peers asynchronously. The server must wait at least five minutes before sharing, but it may wait longer. In this way, the server acts as a buffer, absorbing and "compacting" rapid PUTs.

      This is interesting. I could imagine wanting a more instant version of a tiled view of those I follow -- back when I was on Twitter, there was something cool about the implicit group liveblog of e.g. a major news event. I would have liked to be able to see it all tiled out in one view.

    3. Simple. This means the protocol is easy to understand and implement, even for a non-expert programmer.

      I'm not sure implementation difficulty is the same thing as simplicity. Gemini also says those things are the same. This is also really tricky to evaluate in a world of libraries and linking; we're not mentally including the difficulty of Ed25519 cryptography in our assessment of the protocol's "simplicity" because we're not absolute loons and would use a library for that... but it's part of the fabric being woven.

    4. Accordingly, a Spring '83 realm is limited to 10 million boards

      My gut desire is always for social media accommodating closer to Darius's 50-ish, Dunbar's tiers. I wonder how allowing for media publishers needs to change those numbers.

    5. display each board in a region with an aspect ratio of either 1:sqrt(2) or sqrt(2):1

      Is this really compatible with "[embracing] the richness, flexibility, and chaos of modern HTML and CSS"? I don't have a big negative opinion about it because fixed display size is how webgardens work, too, but articulating the root of the design decision (to allow things to tile nicely?) could open interesting discussion.

    6. load any images, media, or fonts linked by the board

      sobs

    7. (It also means the protocol doesn't provide any mechanism for replies, likes, favorites, or, indeed, feedback of any kind. Publishers are encouraged to use the full flexibility of HTML to develop their own approaches, inviting readers to respond via email, join a live chat, send a postcard... whatever!)

      This has chafed at the users of Gemini even while its designers seem to be proud of holding the line.

    1. please assume that any- thing I'm not commenting on is an enthusiastic "wow this is cool!"

      Yeah, ditto.

  10. Oct 2021
    1. Author Response:

      Reviewer #1 (Public Review):

      In this manuscript by Gilbert et al., the authors found that MLC1 is required for postnatal maturation of perivascular astrocyte coverage. Through various detailed experiments, they further found that Mlc1 KO mice showed a number of defects, including the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow. The data is well presented, however there are several points that need to be addressed to strengthen the manuscript.

      1) Since many PvAP proteins showed the normal expression after P60 in Mlc1 KO mice, it is also possible that many of the phenotype that the authors presented, such as the reduced VSMC contractility, neurovascular coupling and parenchymal CSF flow, can be recovered after P60. This would be important as well to understand the prime pathological cause of megalencephalic leukoencephalopathy induced by MLC1 deletion.

      Although some protein levels are normal in Mlc1 KO mice, PvAP coverage and gliovascular unit morphology are altered at P60. We also now show (using TEM) that PvAPs are swollen in 1-year-old Mlc1 KO mice (the new Fig. S4) - indicating that edema develops progressively in Mlc1 KO mice. Myelin vacuolation starts at 3 months and progressively worsens (Dubey et al., 2015). Taken as a whole, these data show that MLC is a degenerative disease. Under these conditions, the recovery of gliovascular unit function after P60 is very unlikely.

      All the molecular morphological and functional changes in gliovascular unit described in our manuscript precede myelin degradation, which starts at the age of 3 months in Mlc1 KO mice (Dubey et al., 2015). Moreover, our previous study (Gilbert et al., 2019) demonstrated that MLC1 expression starts around P5 and that the MLC1/GlialCAM complex is only mature at P15. Taken together, these results strongly suggest that gliovascular unit alterations are the primary pathological events in MLC.

      Dubey M, Bugiani M, Ridder MC, Postma NL, Brouwers E, Polder E, Jacobs JG, Baayen JC, Klooster J, Kamermans M, et al. 2015. Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle. Ann Neurol 77: 114-131.10.1002/ana.24307. Gilbert A, Vidal XE, Estevez R, Cohen-Salmon M, and Boulay AC. 2019. Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation. Brain Struct Funct 224: 1267-1278.10.1007/s00429-019-01832-w.

      2) Does CBF get differed only after neuronal stimulation in Mlc1 KO mice? It is unclear whether the basal CBF/neurovascular coupling level is disrupted as well in Mlc KO brains and how this defect is related to the reduced vasoconstriction in these mice.

      The baselines of the functional ultrasound experiments were aligned prior to stimulation. This technique measures the percentage increase in blood flow after neuronal stimulation (here, whisker movement) but does not measure the basal flow and does not enable one to distinguish between an abnormal basal cerebral blood flow (as suggested by the reduction of the arterial diameter) and the loss of vascular contractility - both of which probably contribute to the defect in neurovascular coupling.

      3) The reduced cohesiveness of PvAPs and the associated neuronal fibers to the vessel in Mlc1 KO brains should be validated with additional experimental approach.

      To strengthen our analysis, we now give the results of a parallel quantitative immunofluorescence analysis of purified brain vessels (presented in a new figure, Fig.5). The results show that part of the Aqp4 and NF-M perivascular immunolabeling is absent in the Mlc1 KO. Taken as a whole, our data demonstrate that PvAPs and the associated neuronal fibers (which normally remain attached to brain vessels during mechanical purification) are lost during the purification process in Mlc1 KO mice but not in the WT. In conclusion, the absence of MLC1 reduces the mechanical cohesiveness of PvAPs and the associated neuronal fibers.

      4) The defective polarity of astrocytes should be better described by using other markers other than GFAP. The distribution of Aquaporin4, Cx43 or several glutamate transporters in the specific compartment of astrocytes can be examined.

      GFAP is the marker typically used to analyze the astrocytes’ overall morphology and polarity. Nevertheless, we agree that it is of interest to study the molecular polarity of PvAPs. Indeed, morphological changes in the PvAPs and astrocytes and changes in polarity in Mlc1 KO might all influence the localization of molecules in PvAPs. To address this question, we performed a quantitative stimulated emission depletion (STED) analysis of protein localization in PvAPs. Our results indicate that the perivascular localization of aquaporin 4 was not affected. However, the density and size of Cx43 puncta were greater - indicating that the gap junctions in PvAPs are not organized in the same way in the Mlc1 KO as in the WT. This observation is consistent with our electron microscopy observations of perivascular astrocytic processes stacked on the top of each other and linked by extended gap junctions.

      We have also added results for Kir4.1, a potassium channel that is expressed preferentially in PvAPs. The Kir4.1 expression level in Mlc1 KO was lower at all stages of development, indicating that perivascular potassium homeostasis was probably perturbed. These results are interesting because (i) epilepsy is a significant component of megalencephalic leukoencephalopathy (Dubey et al., 2018; Yalcinkaya et al., 2003), and (ii) Kir4.1 deletion or downregulation is associated with greater susceptibility to epilepsy (Sibille et al., 2014). These points are now discussed.

      Dubey M, Brouwers E, Hamilton EMC, Stiedl O, Bugiani M, Koch H, Kole MHP, Boschert U, Wykes RC, Mansvelder HD, et al. 2018. Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts. Ann Neurol 83: 636- 649.10.1002/ana.25190. Sibille J, Pannasch U, and Rouach N. 2014. Astroglial potassium clearance contributes to short-term plasticity of synaptically evoked currents at the tripartite synapse. J Physiol 592: 87- 102.jphysiol.2013.261735 [pii] 10.1113/jphysiol.2013.261735. Yalcinkaya C, Yuksel A, Comu S, Kilic G, Cokar O, and Dervent A. 2003. Epilepsy in vacuolating megalencephalic leukoencephalopathy with subcortical cysts. Seizure 12: 388-396.10.1016/s1059-1311(02)00350-3.

      5) The authors provide interesting observations such that the formation of perivascular astrocyte coverages is required for the dissociation of the contacts between neuronal components and the vessel during development. The authors need to discuss more about potential regulation and implication of this phenomenon.

      This is indeed a fascinating phenomenon. The postnatal period is also an intense synaptogenic phase in the mouse brain (Chung et al., 2015), during which astrocytes and neurons might compete for the perivascular space. In the absence of MLC1 and thus PvAPs, the neurons might expand into the free space. We now comment on this point.

      Chung WS, Allen NJ, and Eroglu C. 2015. Astrocytes Control Synapse Formation, Function, and Elimination. Cold Spring Harb Perspect Biol 7: a020370.cshperspect.a020370 [pii] 10.1101/cshperspect.a020370.

      6) It is interesting that DOTA-Gd tracer shows different traces in Mlc1 KO brains. However, it is unclear how MLC1 deletion affects glymphatic system. Does the tracer normally enter to the perivascular spaces in Mlc KO brains? Does the tracer leak out more from the perivascular spaces in Mlc1 KO mice? Is the general clearance or drainages of the tracer impaired in Mlc1 KO mice? Would these defects be originated by the reduced perivascular astrocyte coverage or the reduced vasoconstriction itself?

      Paravascular transport (as revealed by the injection of a tracer into the CSF) depends mainly on dispersion of the tracer in the subarachnoid space (SAS), the cisternae, and the parenchyma (including the interstitial and perivascular spaces). The uneven, slow dispersion of the tracer within the SAS (compared with dispersion in the blood) means that the tracer’s kinetics in the parenchyma are regiondependent. These differences can be accentuated by regional differences in the anatomy of the brain’s vasculature, i.e. the presence or absence of a perivascular space and the vessel’s topology. Lastly, the amount of DOTA-Gd available for diffusion within the parenchyma depends directly on its local concentration in the SAS. This can be seen on our contrast concentration maps (see Fig. 8), where the highest DOTA-Gd concentrations are found near the injection site (the cisterna magna), in line with previous reports (Iliff et al., 2012). In the Mlc1 KO model, dispersion of DOTA-Gd is presumably affected in the SAS and the parenchyma.

      With regard to tracer dispersion in the SAS and the cisternae, our anatomical MRI showed that the brain volume is greater in the Mlc1 KO mouse than in the WT (see Fig. 1). These variations in the geometry of the SAS may account for much of the difference between the Mlc1 KO mice and WT mice. Although tracer concentrations appear to be similar in the cerebellum (close to the injection site), they are much lower in the more distant septal area of Mlc1 KO mice - suggesting that tracer transport within the SAS is restricted.

      With regard to parenchymal dispersion, we showed that MLC1 is essential for the position of the astrocytes’ perivascular endfeet. Thus, in Mlc1 KO mice, the formation of the perivascular space (as a conduit for solute distribution) is likely to be deficient. This aspect is revealed by the slope of the tracer’s concentration-time curve, which indicate slower kinetics in Mlc1 KO mice; this might be due to poor integrity of the perivascular space. The higher volume of fluid in the Mlc1 KO parenchyma (reflected by the increased apparent diffusion coefficient (ADC); Fig. 1 and S1) might also be involved in this phenotype.

      The heart beat is the main driver of CSF circulation in the perivascular space (Iliff et al., 2013). The heart rate is very rapid and so the heart exerts a much greater driving force on the CSF than the vasodilation of the vessels induced by neuronal activity. Alterations in vascular contractility observed in Mlc1 KO mice might be involved in the impaired CSF flux but this is unlikely.

      All these points are now discussed in the revised version of the manuscript.

      Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, and Benveniste H. 2013. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. Journal of Clinical Investigation 123: 1299-1309.10.1172/jci67677. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, et al. 2012. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 4:147ra111.10.1126/scitranslmed.3003748.

      Reviewer #2 (Public Review):

      This very interesting manuscript by Gilbert and colleagues uncovers that the astrocyte specific membrane protein MLC1, the mutation of which causes a rare disease called megalencephalic leukoencephalopathy with subcortical Cysts (MLC), plays a fundamental role in the postnatal development of the gliovascular unit and the organization of the perivascular astrocyte processes, in particular. To reach this conclusion, the authors used an elegant multiscale approach including in vivo MRI, in vivo functional ultrasound, ex vivo analysis of vascular constriction, anatomical approaches at the light and electron microscopic level, and molecular characterization of the gliovascular unit from isolated microvessels. The manuscript is very well-written although it uses too many (unnecessary) abbreviations, which prevents a fluid reading of the manuscript, results are well illustrated and convincing and the discussion is reasonable.

      I have a major concern regarding the results reported in Figure 4D, which seem somewhat contradictory to those shown in Figure 6A-F. Indeed, the authors report in Figure 4D that there is less Neurofilament-M protein around isolated microvessels in MLC1 KO mice, whereas Figure 6A-F shows that these animals have more neuronal processes in contact with the vessels than in wiltypes. How can the authors explain this?

      The two situations are not comparable. On one hand, we observed the structure of the gliovascular unit in situ in fixed tissues. On the other, we mechanically purified microvessels. The detachment of astrocytic processes and associated neuronal fibers (linked to the mechanical dissociation of microvessels in the Mlc1 KO mouse) was clearly not counterbalanced by the presence of neuronal fibers contacting the vessels.

  11. Aug 2020
    1. 6.2.15. WIRE ROPE When power source and load are located at extreme distances from one another, or loads are very large, the use of wire rope is suggested. Design and use decisions pertaining to wire ropes rest with the user, but manufacturers generally will help users toward appropriate choices. The following material, based on the Committee of Wire Rope Producers, "Wire Rope User's Manual," current edition, may be used as an initial guide in selecting a rope.

      Wire rope is composed of (1) wires to form a strand, (2) strands wound helically around a core, and (3) a core. Classification of wire ropes is made by giving the number of strands, number of minor strands in a major strand (if any), and nominal number of wires per strand. For example 6 × 7 rope means 6 strands with a nominal 7 wires per strand (in this case no minor strands, hence no middle number). A nominal value simply represents a range. A nominal value of 7 can mean anywhere from 3 to 14, of which no more than 9 are outside wires. A full rope description will also include length, size (diameter), whether wire is preformed or not prior to winding, direction of lay (right or left, indicating the direction in which strands are laid around the core), grade of rope (which reflects wire strength), and core. The most widely used classifications are: 6 × 7, 6 × 19, 6 × 37, 6 × 61, 6 × 91, 6 × 127, 8 × 19, 18 × 7, 19 × 7. Some special constructions are: 3 × 7 (guardrail rope); 3 × 19 (slusher); 6 × 12 (running rope); 6 × 24 and 6 × 30 (hawsers); 6 × 42 and 6 × 6 × 7 (tiller rope); 6 × 3 × 19 (spring lay); 5 × 19 and 6 × 19 (marlin clad); 6 × 25B, 6 × 27H, and 6 × 30G (flattened strand). The diameter of a rope is the circle which just contains the rope. The right-regular lay (in which the wire is twisted in one direction to form the strands and the strands are twisted in the opposite direction to form the rope) is most common. Regular-lay ropes do not kink or untwist and handle easily. Lang-lay ropes (in which wires and strands are twisted in the same direction) are more resistant to abrasive wear and fatigue failure.

      Cross sections of some commonly used wire rope are shown in Fig. 6.2.123. Figure 6.2.124 shows rotation-resistant ropes, and Fig. 6.2.125 shows some special-purpose constructions.

      Figure 6.2.123 Cross sections of some commonly used wire rope construction. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/d03c7ddb475211da/158e7be668be00fdf011bdadd2ef3b2300f542e92687c6e02a998fc06e5a48d6/06x02_123.png Open in new tab Share Figure 6.2.124 Cross section of some rotation-resistant wire ropes. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/e19ef67be879c4d0/9ab225782b323010f79e566c12a458512274145d6877d8209774da72c34eda94/06x02_124.png Open in new tab Share Figure 6.2.125 Some special constructions. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/8627b14662f8a537/bb284940d47ada0df68e2027b63f42e8b5f813ef1a6eb57a1dc9e1bdcb726b5b/06x02_125.png Open in new tab Share The core provides support for the strands under normal bending and loading. Core materials include fibers (hard vegetable or synthetic) or steel (either a strand or an independent wire rope). Most common core designations are: fiber core (FC), independent wire-rope core (IWRC), and wire-strand core (WSC). Lubricated fiber cores can provide lubrication to the wire, but add no real strength and cannot be used in high temperature environments. Wire-strand or wire-rope cores add from 7 to 10 percent to strength, but under nonstationary usage tend to wear from interface friction with the outside strands. Great flexibility can be achieved when wire rope is used as strands. Such construction is very pliable and friction resistant. Some manufacturers will provide plastic coatings (nylon, Teflon, vinyl, etc.) upon request. Such coatings help provide resistance to abrasion, corrosion, and loss of lubricant. Crushing refers to rope damage caused by excessive pressures against drum or sheave, improper groove size, and multiple layers on drum or sheave. Consult wire rope manufacturers in doubtful situations.

      Wire-rope materials and their strengths are reflected as grades. These are: traction steel (TS), mild plow steel (MPS), plow steel (PS), improved plow steel (IPS), and extra improved plow (EIP). The plow steel strength curve forms the basis for calculating the strength of all steel rope wires. American manufacturers use color coding on their ropes to identify particular grades.

      The grades most commonly available and tabulated are IPS and EIP. Two specialized categories, where selection requires extraordinary attention, are elevator and rotation-resistant ropes.

      Elevator rope can be obtained in four principal grades: iron, traction steel, high-strength steel, and extra-high-strength steel.

      Bronze rope has limited use; iron rope is used mostly for older existing equipment.

      6.2.15.1. Selection of Wire Rope Appraisal of the following is the key to choosing the rope best suited to the job: resistance to breaking, resistance to bending fatigue, resistance to vibrational fatigue, resistance to abrasion, resistance to crushing, and reserve strength. Along with these must be an appropriate choice of safety factor, which in turn requires careful consideration of all loads, acceleration-deceleration, shocks, rope speed, rope attachments, sheave arrangements as well as their number and size, corrosive and/or abrasive environment, length of rope, etc. An approximate selection formula can be written as:

      DSL

      (NS) K b K sf where DSL (demanded static load) = known or dead load plus additional loads caused by sudden starts or stops, shocks, bearing friction, etc., tons; NS (nominal strength) = published test strengths, tons (see Table 6.2.65); Kb = a factor to account for the reduction in nominal strength due to bending when a rope passes over a curved surface such as a stationary sheave or pin (see Fig. 6.2.126); Ksf = safety factor. (For average operation use Ksf = 5. If there is danger to human life or other critical situations, use 8 ≤ Ksf ≤ 12. For instance, for elevators moving at 50 ft/min, Ksf = 8, while for those moving at 1,500 ft/min, Ksf = 12.)

      Table 6.2.65 Selected Values of Nominal Strengths of Wire Rope Classification

      Nominal diameter

      Fiber core

      IWRC

      Approximate mass

      Nominal strength IPS

      Approximate mass

      Nominal strength

      IPS

      EIP

      in

      mm

      lb/ft

      kg/m

      tons

      t

      lb/ft

      kg/m

      tons

      t

      tons

      t

      Source: "Wire Rope User's Manual," AISI, adapted by permission.

      6 × 7 Bright (uncoated)

      ¼

      6.4

      0.09

      0.14

      2.64

      2.4

      0.10

      0.15

      2.84

      2.58

      3 / 8

      9.5

      0.21

      0.31

      5.86

      5.32

      0.23

      0.34

      6.30

      5.72

      ½

      13

      0.38

      0.57

      10.3

      9.35

      0.42

      0.63

      11.1

      10.1

      16

      0.59

      0.88

      15.9

      14.4

      0.65

      0.97

      17.1

      15.5

      22

      1.15

      1.71

      30.7

      27.9

      1.27

      1.89

      33.0

      29.9

      1⅛

      29

      1.90

      2.83

      49.8

      45.2

      2.09

      3.11

      53.5

      48.5

      1 3 / 8

      35

      2.82

      4.23

      73.1

      66.3

      3.12

      4.64

      78.6

      71.3

      6 × 19 Bright (uncoated)

      ¼

      6.4

      0.11

      0.16

      2.74

      2.49

      0.12

      0.17

      2.94

      2.67

      3.40

      3.08

      3 / 8

      9.5

      0.24

      0.35

      6.10

      5.53

      0.26

      0.39

      6.56

      5.95

      7.55

      6.85

      ½

      13

      0.42

      0.63

      10.7

      9.71

      0.46

      0.68

      11.5

      10.4

      13.3

      12.1

      16

      0.66

      0.98

      16.7

      15.1

      0.72

      1.07

      17.7

      16.2

      20.6

      18.7

      22

      1.29

      1.92

      32.2

      29.2

      1.42

      2.11

      34.6

      31.4

      39.8

      36.1

      1⅛

      29

      2.13

      3.17

      52.6

      47.7

      2.34

      3.48

      56.5

      51.3

      65.0

      59.0

      1 3 / 8

      35

      3.18

      4.73

      77.7

      70.5

      3.5

      5.21

      83.5

      75.7

      96.0

      87.1

      1⅝

      42

      4.44

      6.61

      107

      97.1

      4.88

      7.26

      115

      104

      132

      120

      1⅞

      48

      5.91

      8.8

      141

      128

      6.5

      9.67

      152

      138

      174

      158

      2⅛

      54

      7.59

      11.3

      179

      162

      8.35

      12.4

      192

      174

      221

      200

      2 3 / 8

      60

      9.48

      14.1

      222

      201

      10.4

      15.5

      239

      217

      274

      249

      2⅝

      67

      11.6

      17.3

      268

      243

      12.8

      19.0

      288

      261

      331

      300

      6 × 37 Bright (uncoated)

      ¼

      6.4

      0.11

      0.16

      2.74

      2.49

      0.12

      0.17

      2.94

      2.67

      3.4

      3.08

      3 / 8

      9.5

      0.24

      0.35

      6.10

      5.53

      0.26

      0.39

      6.56

      5.95

      7.55

      6.85

      ½

      13

      0.42

      0.63

      10.7

      9.71

      0.46

      0.68

      11.5

      10.4

      13.3

      12.1

      16

      0.66

      0.98

      16.7

      15.1

      0.72

      1.07

      17.9

      16.2

      20.6

      18.7

      22

      1.29

      1.92

      32.2

      29.2

      1.42

      2.11

      34.6

      31.4

      39.5

      36.1

      1⅛

      29

      2.13

      3.17

      52.6

      47.7

      2.34

      3.48

      56.5

      51.3

      65.0

      59.0

      1 3 / 8

      35

      3.18

      4.73

      77.7

      70.5

      3.50

      5.21

      83.5

      75.7

      96.0

      87.1

      1⅝

      42

      4.44

      6.61

      107

      97.1

      4.88

      7.26

      115

      104

      132

      120

      1⅞

      48

      5.91

      8.8

      141

      128

      6.5

      9.67

      152

      138

      174

      158

      2⅛

      54

      7.59

      11.3

      179

      162

      8.35

      12.4

      192

      174

      221

      200

      2 3 / 8

      60

      9.48

      14.1

      222

      201

      10.4

      15.5

      239

      217

      274

      249

      2⅞

      67

      11.6

      17.3

      268

      243

      12.8

      19.0

      288

      261

      331

      300

      3⅛

      74

      13.9

      20.7

      317

      287

      15.3

      22.8

      341

      309

      392

      356

      80

      16.4

      24.4

      371

      336

      18.0

      26.8

      399

      362

      458

      415

      Open in new tab Share Figure 6.2.126 Values of Kbend vs. D/d ratios (D = sheave diameter, d = rope diameter), based on standard test data for 6 × 9 and 6 × 17 class ropes. (Compiled from "Wire Rope User's Manual," AISI, by permission.) Interactive Graph Values of Kbend vs. D/d ratios (D = sheave diameter, d = rope diameter), based on standard test data for 6 × 9 and 6 × 17 class ropes. (Compiled from "Wire Rope User's Manual," AISI, by permission.) Click on the graph to launch interactivity or enter values below. D/d ratio Kb Open in new tab Share Having made a tentative selection of a rope based on the demanded static load, one considers next the wear life of the rope. A loaded rope bent over a sheave stretches elastically and so rubs against the sheave, causing wear of both members. Drum or sheave size is of paramount importance at this point.

      6.2.15.2. Sizing of Drums or Sheaves Diameters of drums or sheaves in wire rope applications are controlled by two main considerations: (1) the radial pressure between rope and groove and (2) degree of curvature imposed on the rope by the drum or sheave size.

      Radial pressures can be calculated from p = 2T/(Dd), where p = unit radial pressure, lb/in2; T = rope load, lb; D = tread diameter of drum or sheave, in; d = nominal diameter of rope, in. Table 6.2.66 lists suggested allowable radial bearing pressures of ropes on various sheave materials.

      Table 6.2.66 Suggested Allowable Radial Bearing Pressures of Ropes on Various Sheave Materials Material

      Regular lay rope, lb/in2

      Lang lay rope, lb/in2

      Flattened strand lang lay, lb/in2

      Remarks

      6 × 7

      6 × 19

      6 × 37

      8 × 19

      6 × 7

      6 × 19

      6 × 37

      Source: "Wire Rope User's Manual," AISI, reproduced by permission.

      Wood

      150

      250

      300

      350

      165

      275

      330

      400

      On end grain of beech, hickory, gum.

      Cast iron

      300

      480

      585

      680

      350

      550

      660

      800

      Based on minimum Brinell hardness of 125.

      Carbon-steel casting

      550

      900

      1,075

      1,260

      600

      1,000

      1,180

      1,450

      30-40 carbon. Based on minimum Brinell hardness of 160.

      Chilled cast iron

      650

      1,100

      1,325

      1,550

      715

      1,210

      1,450

      1,780

      Not advised unless surface is uniform in hardness.

      Manganese steel

      1,470

      2,400

      3,000

      3,500

      1,650

      2,750

      3,300

      4,000

      Grooves must be ground and sheaves balanced for high-speed service.

      Open in new tab Share All wire ropes operating over drums or sheaves are subjected to cyclical stresses, causing shortened rope life because of fatigue. Fatigue resistance or relative service life is a function of the ratio D/d. Adverse effects also arise out of relative motion between strands during passage around the drum or sheave. Additional adverse effects can be traced to poor match between rope and groove size, and to lack of rope lubrication. Table 6.2.67 lists suggested and minimum sheave and drum ratios for various rope construction. Table 6.2.68 lists relative bending life factors; Figure 6.2.127 shows a plot of relative rope service life versus D/d. Table 6.2.69 lists minimum drum (sheave) groove dimensions. Periodic groove inspection is recommended, and worn or corrugated grooves should be re-machined or the drum replaced, depending on severity of damage.

      Table 6.2.67 Sheave and Drum Ratios Construction* †

      Suggested

      Minimum

      • WS—Warrington Seale; FWS—Filler Wire Seale; SFW—Seale Filler Wire; SWS—Seale Warrington Seale; S—Seale; FW—Filler Wire.

      † D = tread diameter of sheave; d = nominal diameter of rope. To find any tread diameter from this table, the diameter for the rope construction to be used is multiplied by its nominal diameter d. For example, the minimum sheave tread diameter for a ½-in 6 × 21 FW rope would be ½ in (nominal diameter) × 30 (minimum ratio), or 15 in.

      Note: These values are for reasonable service. Other values are permitted by various standards such as ANSI, API, PCSA, HMI, CMAA, etc. Similar values affect rope life.

      Source: "Wire Rope User's Manual," AISI, reproduced by permission.

      6 × 7

      72

      42

      19 × 7 or 18 × 7 Rotation-resistant

      51

      34

      6 × 19 S

      51

      34

      6 × 25 B flattened strand

      45

      30

      6 × 27 H flattened strand

      45

      30

      6 × 30 G flattened strand

      45

      30

      6 × 21 FW

      45

      30

      6 × 26 WS

      45

      30

      6 × 25 FW

      39

      26

      6 × 31 WS

      39

      26

      6 × 37 SFW

      39

      26

      6 × 36 WS

      35

      23

      6 × 43 FWS

      35

      23

      6 × 41 WS

      32

      21

      6 × 41 SFW

      32

      21

      6 × 49 SWS

      32

      21

      6 × 46 SFW

      28

      18

      6 × 46 WS

      28

      18

      8 × 19 S

      41

      27

      8 × 25 FW

      32

      21

      6 × 42 Tiller

      21

      14

      Open in new tab Download data Share Table 6.2.68 Relative Bending Life Factors Rope construction

      Factor

      Rope construction

      Factor

      Source: "Wire Rope User's Manual," AISI, reproduced by permission.

      6 × 7

      0.61

      6 × 36 WS

      1.16

      19 × 7 or 18 × 7

      0.67

      6 × 43 FWS

      1.16

      Rotation-resistant

      0.81

      6 × 41 WS

      1.30

      6 × 19 S

      0.90

      6 × 41 SFW

      1.30

      6 × 25 B flattened strand

      0.90

      6 × 49 SWS

      1.30

      6 × 27 H flattened strand

      0.90

      6 × 43 FW (2 op)

      1.41

      6 × 30 G flattened strand

      0.89

      6 × 46 SFW

      1.41

      6 × 21 FW

      0.89

      6 × 46 WS

      1.41

      6 × 26 WS

      1.00

      8 × 19 S

      1.00

      6 × 25 FW

      1.00

      8 × 25 FW

      1.25

      6 × 31 WS

      1.00

      6 × 42 Tiller

      2.00

      6 × 37 SFW

      Open in new tab Download data Share Figure 6.2.127 Service life curves for various D/d ratios. Note that this curve takes into account only bending and tensile stresses. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) Interactive Graph Service life curves for various D/d ratios. Note that this curve takes into account only bending and tensile stresses. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) Click on the graph to launch interactivity or enter values below. D/d ratio Relative rope service life Open in new tab Share Table 6.2.69 Minimum Sheave- and Drum-Groove Dimensions* Nominal rope diameter

      Groove radius

      New

      Worn

      in

      nm

      in

      mm

      in

      mm

      • Values given are applicable to grooves in sheaves and drums; they are not generally suitable for pitch design since this may involve other factors. Further, the dimensions do not apply to traction-

      type elevators; in this circumstance, drum- and sheave-groove tolerances should conform to the elevator manufacturer's specifications. Modern drum design embraces extensive considerations beyond the scope of this publication. It should also be noted that dram grooves are now produced with a number of oversize dimensions and pitches applicable to certain service requirements.

      Source: "Wire Rope User's Manual," AISI, reproduced by permission.

      ¼

      6.4

      0.135

      3.43

      .129

      3.28

      5 / 16

      8.0

      0.167

      4.24

      .160

      4.06

      3 / 8

      9.5

      0.201

      5.11

      .190

      4.83

      7 / 16

      11

      0.234

      5.94

      .220

      5.59

      ½

      13

      0.271

      6.88

      .256

      6.50

      9 / 16

      14.5

      0.303

      7.70

      .288

      7.32

      5 / 8

      16

      0.334

      8.48

      .320

      8.13

      3 / 4

      19

      0.401

      10.19

      .380

      9.65

      7 / 8

      22

      0.468

      11.89

      .440

      11.18

      1

      26

      0.543

      13.79

      .513

      13.03

      1⅛

      29

      0.605

      15.37

      .577

      14.66

      32

      0.669

      16.99

      .639

      16.23

      1 3 / 8

      35

      0.736

      18.69

      .699

      17.75

      38

      0.803

      20.40

      .759

      19.28

      1⅝

      42

      0.876

      22.25

      .833

      21.16

      45

      0.939

      23.85

      .897

      22.78

      1⅞

      48

      1.003

      25.48

      .959

      24.36

      2

      52

      1.085

      27.56

      1.025

      26.04

      2⅛

      54

      1.137

      28.88

      1.079

      27.41

      58

      1.210

      30.73

      1.153

      29.29

      2 3 / 8

      60

      1.271

      32.28

      1.199

      30.45

      64

      1.338

      33.99

      1.279

      32.49

      2⅝

      67

      1.404

      35.66

      1.339

      34.01

      71

      1.481

      37.62

      1.409

      35.79

      2⅞

      74

      1.544

      39.22

      1.473

      37.41

      3

      77

      1.607

      40.82

      1.538

      39.07

      3⅛

      80

      1.664

      42.27

      1.598

      40.59

      83

      1.731

      43.97

      1.658

      42.11

      3 3 / 8

      87

      1.807

      45.90

      1.730

      43.94

      90

      1.869

      47.47

      1.794

      45.57

      96

      1.997

      50.72

      1.918

      48.72

      4

      103

      2.139

      54.33

      2.050

      52.07

      109

      2.264

      57.51

      2.178

      55.32

      115

      2.396

      60.86

      2.298

      58.37

      122

      2.534

      64.36

      2.434

      61.82

      5

      128

      2.663

      67.64

      2.557

      64.95

      135

      2.804

      71.22

      2.691

      68.35

      141

      2.929

      74.40

      2.817

      71.55

      148

      3.074

      78.08

      2.947

      74.85

      6

      154

      3.198

      81.23

      3.075

      78.11

      Open in new tab Share Seizing and Cutting Wire Rope Before a wire rope is cut, seizings (bindings) must be applied on either side of the cut to prevent rope distortion and flattening or loosened strands. Normally, for preformed ropes, one seizing on each side of the cut is sufficient, but for ropes that are not preformed a minimum of two seizings on each side is recommended, and these should be spaced six rope diameters apart (see Fig. 6.2.128). Seizings should be made of soft or annealed wire or strand, and the width of the seizing should never be less than the diameter of the rope being seized. Table 6.2.70 lists suggested seizing wire diameters.

      Figure 6.2.128 Seizings. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/b5689cae67236644/dabbb5c8ab916a67588e9e15144fa3607cde3a92171221b8c915b2690ccc4448/06x02_128.png Open in new tab Share Table 6.2.70 Seizing* Rope diameter

      Suggested seizing wire diameter†

      in

      mm

      in

      mm

      • Length of the seizing should not be less than the rope diameter.

      † The diameter of seizing wire for elevator ropes is usually somewhat smaller than that shown in this table. Consult the wire rope manufacturer for specific size recommendations. Soft annealed seizing strand may also be used.

      Source: "Wire Rope User's Manual," AISI, reproduced by permission.

      ⅛- 5 / 16

      3.5-8.0

      0.032

      0.813

      3 /

      8

      9 / 16

      9.4-14.5

      0.048

      1.21

      ⅝- 15 / 16

      16.0-24.0

      0.063

      1.60

      1-1 5 / 16

      26.0-33.0

      0.080

      2.03

      1 3 / 8 -1 11 / 16

      35.0-43.0

      0.104

      2.64

      1¾ and larger

      45.0 and larger

      0.124

      3.15

      Open in new tab Share Wire Rope Fittings or Terminations End terminations allow forces to be transferred from rope to machine, or load to rope, etc. Figure 6.2.129 illustrates the most commonly used end fittings or terminations. Not all terminations will develop full strength. In fact, if all of the rope elements are not held securely, the individual strands will sustain unequal loads causing unequal wear among them, thus shortening the effective rope service life. Socketing allows an end fitting which reduces the chances of unequal strand loading.

      Figure 6.2.129 End fittings, or terminations, showing the six most commonly used. (Reproduced from "Wire Rope User's Manual, " AISI, by permission.) binary://mheaeworks/93cbb5f808260c54/0b81d52ad105f0752c71c8e820d154a2b2caedd00b267e91c7d74013bc0b3bbc/06x02_129.png Open in new tab Share Wire rope manufacturers have developed a recommended procedure for socketing. A tight wire serving band is placed where the socket base will be, and the wires are unlaid, straightened, and "broomed" out. Fiber core is cut close to the serving band and removed, wires are cleaned with a solvent such as SC-methyl chloroform, and brushed to remove dirt and grease. If additional cleaning is done with muriatic acid this must be followed by a neutralizing rinse (if possible, ultrasonic cleaning is preferred). The wires are dipped in flux, the socket is positioned, zinc (spelter) is poured and allowed to set, the serving band is removed, and the rope lubricated.

      A somewhat similar procedure is used in thermoset resin socketing.

      Socketed terminations generally are able to develop 100 percent of nominal strength.

  12. Jul 2020
    1. dates Education City, an area devoted to research and education. The city was host to the first ministerial-level meeting of the Doha Development Round of World Trade Organization negotiations. It was also selected as host city of a number of sporting events, including the 2006 Asian Games, the 2011 Pan Arab Games and most of the games at the 2011 AFC Asian Cup. In December 2011, the World Petroleum Council held the 20th World Petroleum Conference in Doha.[4] Additionally, the city hosted the 2012 UNFCCC Climate Negotiations and is set to host many of the venues for the 2022 FIFA World Cup.[5] The city has also hosted the 140th Inter-Parliamentary Union Assembly in April 2019. Contents 1 Etymology 2 History 2.1 Establishment of Al Bidda 2.2 Formation of Doha 2.3 Arrival of Al Thani 2.4 20th century 2.4.1 Lorimer report (1908) 2.4.2 British protectorate (1916–1971) 2.5 Post-independence 3 Geography 3.1 Climate 4 Demographics 4.1 Ethnicity and languages 4.2 Religion 5 Administration 5.1 Districts 6 Economy 7 Infrastructure 7.1 Architecture 7.2 Atmosphere 7.3 Planned communities 8 Transportation 8.1 Roads 8.2 Rail 8.3 Air 9 Education 10 Sports 10.1 Football 10.2 Basketball 10.3 Volleyball 10.4 Other sports 10.5 Stadiums and sport complexes 11 Culture 11.1 Arts 11.2 Cinema 11.3 Media 11.4 Theatre 12 International relations 13 Gallery 14 See also 15 References 16 External links Etymology[edit] According to the Ministry of Municipality and Environment, the name "Doha" originated from the Arabic term dohat, meaning "roundness"—a reference to the rounded bays surrounding the area's coastline.[6] History[edit] See also: Timeline of Doha and Al Bidda A satellite view of Doha on the East coast of Qatar. As with most world cities, Doha developed on the water front around the Souq Waqif area today. It gradually spread out in a radial pattern with the use of ring roads. Establishment of Al Bidda[edit] The city of Doha was formed seceding from another local settlement known as Al Bidda. The earliest documented mention of Al Bidda was made in 1681, by the Carmelite Convent, in an account which chronicles several settlements in Qatar. In the record, the ruler and a fort in the confines of Al Bidda are alluded to.[7][8] Carsten Niebuhr, a German explorer who visited the Arabian Peninsula, created one of the first maps to depict the settlement in 1765 in which he labelled it as 'Guttur'.[7][9] David Seaton, a British political resident in Muscat, wrote the first English record of Al Bidda in 1801. He refers to the town as 'Bedih' and describes the geography and defensive structures in the area.[10] He stated that the town had recently been settled by the Sudan tribe (singular Al-Suwaidi), whom he considered to be pirates. Seaton attempted to bombard the town with his warship, but returned to Muscat upon finding that the waters were too shallow to position his warship within striking distance.[11][12] In 1820, British surveyor R. H. Colebrook, who visited Al Bidda, remarked on the recent depopulation of the town. He wrote:[11][13] .mw-parser-output .templatequote{overflow:hidden;margin:1em 0;padding:0 40px}.mw-parser-output .templatequote .templatequotecite{line-height:1.5em;text-align:left;padding-left:1.6em;margin-top:0}Guttur – Or Ul Budee [Al‐Bidda], once a considerable town, is protected by two square Ghurries [forts] near the sea shore; but containing no fresh water they are incapable of defence except against sudden incursions of Bedouins, another Ghurry is situated two miles inland and has fresh water with it. This could contain two hundred men. There are remaining at Ul Budee about 250 men, but the original inhabitants, who may be expected to return from Bahrein, will augment them to 900 or 1,000 men, and if the Doasir tribe, who frequent the place as divers, again settle in it, from 600 to 800 men. The same year, an agreement known as the General Maritime Treaty was signed between the East India Company and the sheikhs of several Persian Gulf settlements (some of which were later known as the Trucial Coast). It acknowledged British authority in the Persian Gulf and sought to end piracy and the slave trade. Bahrain became a party to the treaty, and it was assumed that Qatar, perceived as a dependency of Bahrain by the British, was also a party to it.[14] Qatar, however, was not asked to fly the prescribed Trucial flag.[15] As punishment for alleged piracy committed by the inhabitants of Al Bidda and breach of treaty, an East India Company vessel bombarded the town in 1821. They razed the town, forcing between 300 and 400 natives to flee and temporarily take shelter on the islands between the Qatar and the Trucial Coast.[16] Formation of Doha[edit] Doha was founded in the vicinity of Al Bidda sometime during the 1820s.[17] In January 1823, political resident John MacLeod visited Al Bidda to meet with the ruler and initial founder of Doha, Buhur bin Jubrun, who was also the chief of the Al-Buainain tribe.[17][18] MacLeod noted that Al Bidda was the only substantial trading port in the peninsula during this time. Following the founding of Doha, written records often conflated Al Bidda and Doha due to the extremely close proximity of the two settlements.[17] Later that year, Lt. Guy and Lt. Brucks mapped and wrote a description of the two settlements. Despite being mapped as two separate entities, they were referred to under the collective name of Al Bidda in the written description.[19][20] Al Bidda: View from the bay, 1823 In 1828, Mohammed bin Khamis, a prominent member of the Al-Buainain tribe and successor of Buhur bin Jubrun as chief of Al Bidda, was embroiled in controversy. He had murdered a native of Bahrain, prompting the Al Khalifa sheikh to imprison him. In response, the Al-Buainain tribe revolted, provoking the Al Khalifa to destroy the tribe's fort and evict them to Fuwayrit and Ar Ru'ays. This incident allowed the Al Khalifa additional jurisdiction over the town.[21][22] With essentially no effective ruler, Al Bidda and Doha became a sanctuary for pirates and outlaws.[23] ‘Trigonometrical plan of the harbour of El Biddah on the Arabian side of the Persian Gulf’, 1823 In November 1839, an outlaw from Abu Dhabi named Ghuleta took refuge in Al Bidda, evoking a harsh response from the British. A. H. Nott, a British naval commander, demanded that Salemin bin Nasir Al-Suwaidi, chief of the Sudan tribe (Suwaidi) in Al Bidda, take Ghuleta into custody and warned him of consequences in the case of non-compliance. Al-Suwaidi obliged the British request in February 1840 and also arrested the pirate Jasim bin Jabir and his associates. Despite the compliance, the British demanded a fine of 300 German krones in compensation for the damages incurred by pirates off the coast of Al Bidda; namely for the piracies committed by bin Jabir. In February 1841, British naval squadrons arrived in Al Bidda and ordered Al-Suwaidi to meet the British demand, threatening consequences if he declined. Al-Suwaidi ultimately declined on the basis that he was uninvolved in bin Jabir's actions. On 26 February, the British fired on Al Bidda, striking a fort and several houses. Al-Suwaidi then paid the fine in full following threats of further action by the British.[23][24] Isa bin Tarif, a powerful tribal chief from the Al Bin Ali tribe, moved to Doha in May 1843. He subsequently evicted the ruling Sudan tribe and installed the Al-Maadeed and Al-Kuwari tribes in positions of power.[25] Bin Tarif had been loyal to the Al Khalifa, however, shortly after the swearing in of a new ruler in Bahrain, bin Tarif grew increasingly suspicious of the ruling Al Khalifa and switched his allegiance to the deposed ruler of Bahrain, Abdullah bin Khalifa, whom he had previously assisted in deposing of. Bin Tarif died in the Battle of Fuwayrit against the ruling family of Bahrain in 1847.[25] Arrival of Al Thani[edit] The Al Thani migrated to Doha from Fuwayrit shortly after Bin Tarif's death in 1847 under the leadership of Mohammed bin Thani.[26][27] In the proceeding years, the Al Thani assumed control of the town. At various times, they swapped allegiances between the two prevailing powers in the area: the Al Khalifa and the Saudis.[26] Plan of Al Bidda Harbour drawn in 1860 indicating the principal settlements and landmarks In 1867, many ships and troops were sent from Bahrain to assault the towns Al Wakrah and Doha over a series of disputes. Abu Dhabi joined on Bahrain's behalf due to the conception that Al Wakrah served as a refuge for fugitives from Oman. Later that year, the combined forces sacked the two Qatari towns with around 2,700 men in what would come to be known as the Qatari–Bahraini War.[28][29] A British record later stated "that the towns of Doha and Wakrah were, at the end of 1867 temporarily blotted out of existence, the houses being dismantled and the inhabitants deported".[30] The joint Bahraini-Abu Dhabi incursion and subsequent Qatari counterattack prompted the British political agent, Colonel Lewis Pelly, to impose a settlement in 1868. Pelly's mission to Bahrain and Qatar and the peace treaty that resulted were milestones in Qatar's history. It implicitly recognized Qatar as a distinct entity independent from Bahrain and explicitly acknowledged the position of Mohammed bin Thani as an important representative of the peninsula's tribes.[31] A part of Doha as seen in January 1904. Most development was low-rise and use of locally available natural materials like rammed earth and palm fronds was common practice. In December 1871, the Ottomans established a presence in the country with 100 of their troops occupying the Musallam fort in Doha. This was accepted by Mohammad bin Thani's son, Jassim Al Thani, who wished to protect Doha from Saudi incursions.[32] The Ottoman commander, Major Ömer Bey, compiled a report on Al Bidda in January 1872, stating that it was an "administrative centre" with around 1,000 houses and 4,000 inhabitants.[33] Disagreement over tribute and interference in internal affairs arose, eventually leading to the Battle of Al Wajbah in March 1893. Al Bidda fort served as the final point of retreat for Ottoman troops. While they were garrisoned in the fort, their corvette fired indiscriminately at the townspeople, killing a number of civilians.[34] The Ottomans eventually surrendered after Jassim Al Thani's troops cut off the town's water supply.[35] An Ottoman report compiled the same year reported that Al Bidda and Doha had a combined population of 6,000 inhabitants, jointly referring to both towns by the name of 'Katar'. Doha was classified as the eastern section of Katar.[33][36] The Ottomans held a passive role in Qatar's politics from the 1890s onward until fully relinquishing control during the beginning of the first World War.[14] 20th century[edit] The city's coastline in 1904 largely highlights the local community which was based on fishing and pearl diving. Pearling had come to play a pivotal commercial role in Doha by the 20th century. The population increased to around 12,000 inhabitants in the first half of the 20th century due to the flourishing pearl trade.[37] A British political resident noted that should the supply of pearls drop, Qatar would 'practically cease to exist'.[38] In 1907, the city accommodated 350 pearling boats with a combined crew size of 6,300 men. By this time, the average prices of pearls had more than doubled since 1877.[39] The pearl market collapsed that year, forcing Jassim Al Thani to sell the country's pearl harvest at half its value. The aftermath of the collapse resulted in the establishment of the country's first custom house in Doha.[38] Lorimer report (1908)[edit] British administrator and historian J. G. Lorimer authored an extensive handbook for British agents in the Persian Gulf entitled Gazetteer of the Persian Gulf in 1908. In it, he gives a comprehensive account of Doha at the time: "Dohah looking northwest", photographed by the Royal Air Force during a reconnaissance of the Qatar Peninsula on 9 May 1934 Generally so styled at the present day, but Bedouins sometimes call it Dohat-al-Qatar; and it seems to have been formerly better known as Bida' (Anglice "Bidder"): it is the chief town of Qatar and is situated on the eastern side of that peninsula, about 63 miles south of its extremity at Ras Rakan and 45 miles north of Khor-al Odaid Harbour. Dohah stands on the south side of a deep bay at the south-western corner of a natural harbour which is about 3 miles in extent and is protected on the north-east and south-east sides by natural reefs. The entrance, less than a mile wide, is from the east between the points of the reefs; it is shallow and somewhat difficult, and vessels of more than 15 feet draught cannot pass. The soundings within the basin vary from 3 to 5 fathoms and are regular: the bottom is white mud or clay. Town site and quarters, — The south-eastern point of the bay is quite low but the land on the western side is stony desert 40 or 50 feet above the level of the sea. The town is built up the slope of some rising ground between these two extremes and consists of 9 Fanqs or quarters, which are given below in their order from the east to the west and north: the total frontage of the place upon the sea is nearly 2 miles.[40] An old district in Doha planned with narrow streets and rough plastered walls gives a glimpe of the city's past. Lorimer goes on to list and describe the districts of Doha, which at the time included the still-existing districts of Al Mirqab, As Salatah, Al Bidda and Rumeilah.[41] Remarking on Doha's appearance, he states: The general appearance of Dohah is unattractive; the lanes are narrow and irregular the houses dingy and small. There are no date palms or other trees, and the only garden is a small one near the fort, kept up by the Turkish garrison.[42] As for Doha's population, Lorimer asserts that "the inhabitants of Dohah are estimated to amount, inclusive of the Turkish military garrison of 350 men, to about 12,000 souls". He qualified this statement with a tabulated overview of the various tribes and ethnic groups living in the town.[42] British protectorate (1916–1971)[edit] In April 1913, the Ottomans agreed to a British request that they withdraw all their troops from Qatar. Ottoman presence in the peninsula ceased, when in August 1915, the Ottoman fort in Al Bidda was evacuated shortly after the start of World War I.[43] One year later, Qatar agreed to be a British protectorate with Doha as its official capital.[44][45] Camels next to Al Koot Fort, built in 1927 by Abdullah bin Jassim Al Thani. Buildings at the time were simple dwellings of one or two rooms, built from mud, stone and coral. Oil concessions in the 1920s and 1930s, and subsequent oil drilling in 1939, heralded the beginning of slow economic and social progress in the country. However, revenues were somewhat diminished due to the devaluation of pearl trade in the Persian Gulf brought on by introduction of the cultured pearl and the Great Depression.[46] The collapse of the pearl trade caused a significant population drop throughout the entire country.[37] It was not until the 1950s and 1960s that the country saw significant monetary returns from oil drilling.[14] A view of Doha in the 1980s showing the Sheraton Hotel (pyramid-like building in the background) in West Bay without any of the high-rises around it Qatar was not long in exploiting the new-found wealth from oil concessions, and slum areas were quickly razed to be replaced by more modern buildings. The first formal boys' school was established in Doha in 1952, followed three years later by the establishment of a girls' school.[47] Historically, Doha had been a commercial port of local significance. However, the shallow water of the bay prevented bigger ships from entering the port until the 1970s, when its deep-water port was completed. Further changes followed with extensive land reclamation, which led to the development of the crescent-shaped bay.[48] From the 1950s to 1970s, the population of Doha grew from around 14,000 inhabitants to over 83,000, with foreign immigrants constituting about two-thirds of the overall population.[49] Post-independence[edit] The Pearl-Qatar at night The Pearl-Qatar is an artificial island spanning nearly four square kilometers. Qatar Petroleum tower, Palm tower B, Tornado tower, Doha tower and Al Jassimya tower seen (Left to Right) in the West Bay area in 2015 Qatar officially declared its independence in 1971, with Doha as its capital city.[3] In 1973, the University of Qatar was opened by emiri decree,[50] and in 1975 the Qatar National Museum opened in what was originally the ruler's palace.[51] During the 1970s, all old neighborhoods in Doha were razed and the inhabitants moved to new suburban developments, such as Al Rayyan, Madinat Khalifa and Al Gharafa. The metropolitan area's population grew from 89,000 in the 1970s to over 434,000 in 1997. Additionally, land policies resulted in the total land area increasing to over 7,100 hectares (about 17,000 acres) by 1995, an increase from 130 hectares in the middle of the 20th century.[52] In 1983, a hotel and conference center was developed at the north end of the Corniche. The 15-storey Sheraton hotel structure in this center would serve as the tallest structure in Doha until the 1990s.[52] In 1993, the Qatar Open became the first major sports event to be hosted in the city.[53] Two years later, Qatar stepped in to host the FIFA World Youth Championship, with all the matches being played in Doha-based stadiums.[54] Developments in Doha's West Bay district have seen an increase in the population density of the area with the construction of several high-rises. A view of a water feature in Sheraton Park with the West Bay skyline in the background. The Al Jazeera Arabic news channel began broadcasting from Doha in 1996.[55] In the late 1990s, the government planned the construction of Education City, a 2,500 hectare Doha-based complex mainly for educational institutes.[56] Since the start of the 21st century, Doha attained significant media attention due to the hosting of several global events and the inauguration of a number of architectural mega-projects.[57] One of the largest projects launched by the government was The Pearl-Qatar, an artificial island off the coast of West Bay, which launched its first district in 2004.[58] In 2006, Doha was selected to host the Asian Games, leading to the development of a 250-hectare sporting complex known as Aspire Zone.[53] During this time, new cultural attractions were constructed in the city, with older ones being restored. In 2006, the government launched a restoration program to preserve Souq Waqif's architectural and historical identity. Parts constructed after the 1950s were demolished whereas older structures were refurbished. The restoration was completed in 2008.[59] Katara Cultural Village was opened in the city in 2010 and has hosted the Doha Tribeca Film Festival since then.[60] The main outcome of the World Trade Organization Ministerial Conference of 2013 was the Trade Facilitation Agreement. The agreement aims to make it easier and cheaper to import and export by improving customs procedures and making rules more transparent. Reducing global trade costs by 1% would increase world-wide income more than USD 40 billion, 65% of which would go to developing countries. The gains from the Trade Facilitation Agreement are expected to be distributed among all countries and regions, with developing landlocked countries benefitting the most.[61] The Trade Facilitation Agreement will enter into force upon its ratification by 2/3 of WTO Members. The EU ratified the agreement in October 2015.[61] In Bali, WTO members also agreed on a series of Doha agriculture and development issues.[61] Geography[edit] See also: Geography of Qatar A view of Doha from the International Space Station in 2010 highlights the rapid development the city underwent since the discovery of oil in the 1960s. Doha is located on the central-east portion of Qatar, bordered by the Persian Gulf on its coast. Its elevation is 10 m (33 ft).[62] Doha is highly urbanized. Land reclamation off the coast has added 400 hectares of land and 30 km of coastline.[63] Half of the 22 km² of surface area which Hamad International Airport was constructed on was reclaimed land.[64] The geology of Doha is primarily composed of weathered unconformity on the top of the Eocene period Dammam Formation, forming dolomitic limestone.[65] The Pearl is a purpose-built artificial island off the coast of Doha, connected to the mainland by a bridge. The Pearl is an artificial island in Doha with a surface area of nearly 400 ha (1,000 acres)[66] The total project has been estimated to cost $15 billion upon completion.[67] Other islands off Doha's coast include Palm Tree Island, Shrao's Island, Al Safliya Island, and Alia Island.[68] In a 2010 survey of Doha's coastal waters conducted by the Qatar Statistics Authority, it was found that its maximum depth was 7.5 meters (25 ft) and minimum depth was 2 meters (6 ft 7 in). Furthermore, the waters had an average pH of 7.83, a salinity of 49.0 psu, an average temperature of 22.7 °C and 5.5 mg/L of dissolved oxygen.[69] Climate[edit] Doha has a hot desert climate (Köppen climate classification BWh) with long, extremely hot summers and short, warm winters. The average high temperatures between May and September surpass 38 °C (100 °F) and often approach 45 °C (113 °F). Humidity is usually the lowest in May and June. Dewpoints can surpass 30 °C (86 °F) in the summer. Throughout the summer, the city averages almost no precipitation, and less than 20 mm (0.79 in) during other months.[70] Rainfall is scarce, at a total of 75 mm (2.95 in) per year, falling on isolated days mostly between October to March. The winter's days are relativity warm while the sun is up and cool during the night. The temperature rarely drops below 7 °C (45 °F).[71] hideClimate data for Doha (1962–2013, extremes 1962–2013) Month Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Year Record high °C (°F) 32.4(90.3) 36.5(97.7) 39.0(102.2) 46.0(114.8) 47.7(117.9) 49.1(120.4) 50.4(122.7) 48.6(119.5) 46.2(115.2) 43.4(110.1) 38.0(100.4) 32.7(90.9) 50.4(122.7) Average high °C (°F) 22.0(71.6) 23.4(74.1) 27.3(81.1) 32.5(90.5) 38.8(101.8) 41.6(106.9) 41.9(107.4) 40.9(105.6) 38.9(102.0) 35.4(95.7) 29.6(85.3) 24.4(75.9) 33.1(91.5) Daily mean °C (°F) 17.8(64.0) 18.9(66.0) 22.3(72.1) 27.1(80.8) 32.5(90.5) 35.1(95.2) 36.1(97.0) 35.5(95.9) 33.3(91.9) 30.0(86.0) 25.0(77.0) 20.0(68.0) 27.8(82.0) Average low °C (°F) 13.5(56.3) 14.4(57.9) 17.3(63.1) 21.4(70.5) 26.1(79.0) 28.5(83.3) 30.2(86.4) 30.0(86.0) 27.7(81.9) 24.6(76.3) 20.4(68.7) 15.6(60.1) 22.5(72.5) Record low °C (°F) 3.8(38.8) 5.0(41.0) 8.2(46.8) 10.5(50.9) 15.2(59.4) 21.0(69.8) 23.5(74.3) 22.4(72.3) 20.3(68.5) 16.6(61.9) 11.8(53.2) 6.4(43.5) 3.8(38.8) Average precipitation mm (inches) 13.2(0.52) 17.1(0.67) 16.1(0.63) 8.7(0.34) 3.6(0.14) 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) 1.1(0.04) 3.3(0.13) 12.1(0.48) 75.2(2.95) Average precipitation days (≥ 1.0 mm) 1.7 2.1 1.8 1.4 0.2 0.0 0.0 0.0 0.0 0.1 0.2 1.3 8.8 Average relative humidity (%) 74 70 63 53 44 41 50 58 62 63 66 74 60 Mean monthly sunshine hours 244.9 224.0 241.8 273.0 325.5 342.0 325.5 328.6 306.0 303.8 276.0 241.8 3,432.9 Mean daily sunshine hours 7.9 8.0 7.8 9.1 10.5 11.4 10.5 10.6 10.2 9.8 9.2 7.8 9.4 Source 1: NOAA[71] Source 2: Qatar Meteorological Department (Climate Normals 1962–2013)[72] Doha mean sea temperature[73] Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 20.5 °C (68.9 °F) 19.1 °C (66.4 °F) 20.9 °C (69.6 °F) 23.7 °C (74.7 °F) 28.2 °C (82.8 °F) 30.9 °C (87.6 °F) 32.8 °C (91.0 °F) 33.9 °C (93.0 °F) 33.1 °C (91.6 °F) 31.0 °C (87.8 °F) 27.4 °C (81.3 °F) 23.1 °C (73.6 °F) Demographics[edit] See also: Demographics of Qatar Historical populationYearPop.±%1820[11]250—    1893[33]6,000+2300.0%1970[74]80,000+1233.3%1986[3] 217,294+171.6%1998[75] 264,009+21.5%2001[76] 299,300+13.4%2004[3] 339,847+13.5%2005[77][78] 400,051+17.7%2010[79] 796,947+99.2%2015[2] 956,457+20.0% Total population of the Doha metropolitan area[80] Year Metro population 1997 434,000[52] 2004 644,000[81] 2008 998,651[82] A significant portion of Qatar's population resides within the confines of Doha and its metropolitan area.[83] The district with the highest population density is the central area of Al Najada, which also accommodates the highest total population in the country. The population density across the greater Doha region ranges from 20,000 people per km² to 25 people per km².[84] Doha witnessed explosive growth rates in population in the first decade of the 21st century, absorbing the majority of the thousands of people then immigrating to Qatar every month.[85]:6 Doha's population currently stands at around one million, with the population of the city more than doubling from 2000 to 2010.[2] Ethnicity and languages[edit] The population of Doha is overwhelmingly composed of expatriates, with Qatari nationals forming a minority. The largest portion of expatriates in Qatar are from South-East and South Asian countries, mainly India, Pakistan, Sri Lanka, Nepal, Philippines, and Bangladesh with large numbers of expatriates also coming from the Levant Arab countries, North Africa, and East Asia. Doha is also home to many expatriates from Europe, North America, South Africa and Australia.[86] A typical bilingual traffic sign in Doha denotes the zone numbers, street names and street numbers of two perpendicular streets. Arabic is the official language of Qatar. English is commonly used as a second language,[87] and a rising lingua franca, especially in commerce.[88] As there is a large expatriate population in Doha, languages such as Malayalam, Tamil, Bengali, Tagalog, Spanish, Sinhala, French, Urdu and Hindi are widely spoken.[86] Registered live births in Doha by nationality[80][89] Year Qatari Non-Qatari Total 2001 2,080 3,619 5,699 2002 1,875 3,657 5,532 2003 2,172 4,027 6,199 2004 2,054 3,760 5,814 2005 1,767 3,899 5,666 2006 1,908 4,116 6,024 2007 1,913 4,708 6,621 2008 1,850 5,283 7,133 2009 2,141 5,979 8,120 2010[90] 1,671 5,919 7,590 2011[91] 1,859 6,580 8,439 In 2004, the Foreign Ownership of Real Estate Law was passed, permitting non-Qatari citizens to buy land in designated areas of Doha, including the West Bay Lagoon, the Qatar Pearl, and the new Lusail City.[57] Prior to this, expatriates were prohibited from owning land in Qatar. Ownership by foreigners in Qatar entitles them to a renewable residency permit, which allows them to live and work in Qatar.[83] Religion[edit] Main article: Religion in Qatar The majority of residents in Doha are Muslim.[92] Catholics account for over 90% of the 150,000 Christian population in Doha.[93] Following decrees by the Emir for the allocation of land to churches, the first Catholic church, Our Lady of the Rosary, was opened in Doha in March 2008. The church structure is discreet and Christian symbols are not displayed on the outside of the building.[94] Several other churches exist in Doha, including the [1] St.Isaac and St. George Greek Orthodox Church of Qatar the Syro-Malabar Church, Malankara Orthodox Church, Mar Thoma Church (affiliated with the Anglicans, but not part of the Communion), CSI Church, Syro-Malankara Church and a Pentecostal church. A majority of mosques are either Muwahhid or Sunni-oriented.[95] Administration[edit] Districts[edit] Main article: List of communities in Doha At the turn of the 20th century, Doha was divided into 9 main districts.[96] In the 2010 census, there were more than 60 districts recorded in Doha Municipality.[97] Some of the districts of Doha include: Qatar's Central Bank is situated in the Al Souq district, close to the waterfront. Al Bidda (البدع) Al Dafna (الدفنة) Al Ghanim (الغانم) Al Markhiya (المرخية) Al Sadd (السد) Al Waab (الوعب) Bin Mahmoud (فريج بن محمود) Madinat Khalifa (مدينة خليفة) Musheireb (مشيرب) Najma (نجمه) Old Airport (المطار القديم) Qutaifiya (القطيفية) Ras Abu Aboud (راس أبو عبود) Rumeilah (الرميلة) Umm Ghuwailina (ام غو يلينه) West Bay (الخليج الغربي) Shortly after Qatar gained independence, many of the districts of old Doha including Al Najada, Al Asmakh and Old Al Hitmi faced gradual decline and as a result much of their historical architecture has been demolished.[98] Instead, the government shifted their focus toward the Doha Bay area, which housed districts such as Al Dafna and West Bay.[98] Economy[edit] See also: Economy of Qatar West Bay serves as the commercial district of Doha and houses offices of many local and global companies. Doha is the economic centre of Qatar. The city is the headquarters of numerous domestic and international organizations, including the country's largest oil and gas companies, Qatar Petroleum, Qatargas and RasGas. Doha's economy is built primarily on the revenue the country has made from its oil and natural gas industries.[99] Doha was included in Fortune's 15 best new cities for business in 2011.[100] Beginning in the late 20th century, the government launched numerous initiatives to diversify the country's economy in order to decrease its dependence on oil and gas resources. Doha International Airport was constructed in a bid to solidify the city's diversification into the tourism industry.[99] This was replaced by Hamad International Airport in 2014. The new airport is almost twice the size of the former and features two of the longest runways in the world.[101] Thirty-nine new hotels were under construction in the city in 2011.[102] Qatar Airways aircraft on the apron of Hamad International Airport As a result of Doha's rapid population boom and increased housing demands, real estate prices rose significantly through 2014.[103] Real estate prices experienced a further spike after Qatar won the rights to host the 2022 FIFA World Cup.[104] Al Asmakh, a Qatari real estate firm, released a report in 2014 which revealed substantial increases in real estate prices following a peak in 2008. Prices increased 5 to 10% in the first quarter of 2014 from the end of 2013.[103][105] A 2015 study conducted by Numbeo, a crowd-sourced database, named Doha as the 10th most expensive city to live in globally.[106] This rate of growth led to the development of planned communities in and around the city.[107] Although the fall in oil prices since 2014 and a diplomatic crisis with Qatar's neighbors slowed growth in the city's population, government spending was increased to maintain the growth in real estate in metropolitan Doha.[108] Expatriate workers remitted $60bn between 2006 and 2012, with 54 percent of the workers' remittances of $60bn routed to Asian countries, followed by Arab nations that accounted for nearly half that volume (28 percent). India was the top destination of the remittances, followed by the Philippines, while the US, Egypt and the neighbouring UAE followed.[109] Remittances in 2014 totaled $11.2 billion, amounting to 5.3% of Qatar's GDP.[110]:45 Infrastructure[edit] See also: List of tallest buildings in Qatar Architecture[edit] Museum of Islamic Art park in the Doha Port area with the West Bay district in the background (across the bay) Most traditional architecture in the Old Doha districts have been demolished to make space for new buildings.[98] As a result, a number of schemes have been taken to preserve the city's cultural and architectural heritage, such as the Qatar Museums Authority's 'Al Turath al Hai' ('living heritage') initiative.[111] Katara Cultural Village is a small village in Doha launched by sheikh Tamim Al Thani to preserve the cultural identity of the country.[112] Doha's Al Dafna area with the high-rises seen on the water front and the villa compounds and other residential areas seen in the background In 2011, more than 50 towers were under construction in Doha,[102] the largest of which was the Doha Convention Center Tower.[113] Constructions were suspended in 2012 following concerns that the tower would impede flight traffic.[114] In 2014, Abdullah Al Attiyah, a senior government official, announced that Qatar would be spending $65bn on new infrastructure projects in upcoming years in preparation for the 2022 World Cup as well as progressing towards its objectives set out in the Qatar National Vision 2030.[115] Atmosphere[edit] Due to excessive heat from the sun during the summer, some Doha-based building companies have implemented various forms of cooling technology to alleviate the extremely torrid climatic conditions. This can include creating optical phenomena such as shadows, as well as more expensive techniques like ventilation, coolants, refrigerants, cryogenics, and dehumidifiers.[116] Discussions regarding temperature control have also been features of various scheduled events involving large crowds.[117] There are other initiatives that attempt to counter the heat by altering working hours, weather alteration methods such as cloud seeding,[118][119] and using whiter and brighter construction materials to increase the albedo effects.[120] Nonetheless, despite these measures, Doha and other areas of Qatar could become uninhabitable for humans due to climate change by the 2070s.[121] Planned communities[edit] One of the largest projects underway in Qatar is Lusail City, a planned community north of Doha which is estimated to be completed by 2020 at a cost of approximately $45bn. It is designed to accommodate 450,000 people.[122] Al Waab City, another planned community under development, is estimated to cost QR15 bn.[123] In addition to housing 8,000 individuals, it will also have shopping malls, educational, and medical facilities.[123] Transportation[edit] Main article: Transportation in Doha Since 2004, Doha has been undergoing a huge expansion to its transportation network, including the addition of new highways, the opening of a new airport in 2014, and the currently ongoing construction of an 85 km metro system. This has all been as a result of Doha's massive growth in a short period of time, which has resulted in congestion on its roads. The first phase of the metro system is expected to be operational by 2019.[124] Roads[edit] Dukhan Highway connects the city of Dukhan on the West coast of the country with the country's capital, Doha. In 2015, the Public Works Authority declared their plan to construct a free-flowing road directly linking Al-Wakrah and Mesaieed to Doha in order to decrease traffic congestion in the city. It is set for completion by 2018.[125] Commutes between Doha and the municipality of Al Khor are currently facilitated by Al Shamal Road and Al Khor Coastal Road, with the latter road running through Al Daayen and the former running through Umm Salal.[126] Doha is linked to the country's western settlements, namely Dukhan, through Dukhan Highway. The Public Works Authority carried out the Dukhan Highway Central Project in 2017 to enhance the road network.[127] Rail[edit] Doha Metro will consist of four lines: the Red Line, the Gold Line, the Blue Line and the Green Line. The Blue Line is expected to be completed in the second phase.[128] Msheireb Station will be the point of intersection for all of the metro lines.[124] Doha International Airport The Red Line (also known as Coast Line) will extend through Doha, running from Al Wakrah to Al Khor. It is separated into two divisions: Red Line North and Red Line South. The former will run from Mushayrib Station to Al Khor City, over a length of 55.7 km. Doha Metro's Green Line will connect Doha to Education City and is also known as the Education Line. Starting in Old Airport, the Gold Line (also known as Historic Line) will end in Al Rayyan and cover a distance of 30.6 km. Lastly, the Blue Line, or City Line, will only cover the city of Doha, and is planned to be circular with a length of 17.5 km.[129] Air[edit] Doha is served by Hamad International Airport which is Qatar's principal international gateway. The airport opened in 2014, replacing Doha International Airport. Education[edit] See also: Education in Qatar, Education City, and List of schools in Qatar Stone steps at Carnegie Mellon University in Qatar, located in Education City Doha is the educational center of the country and contains the highest preponderance of schools and colleges.[74] In 1952, the first formal boys' school was opened in Doha. This was proceeded by the opening of the first formal girls' school three years later.[130] The first university in the state, Qatar University, was opened in 1973.[131] It provided separate faculties for men and women.[132] Education City, a 14 km2 education complex launched by non-profit organization Qatar Foundation, began construction in 2000.[133] It houses eight universities, the country's top high school, and offices for Al Jazeera's children television channel.[133] It is geographically located in Al Rayyan municipality's Al Luqta, Al Gharrafa, Gharrafat Al Rayyan and Al Shagub districts, but falls under the umbrella of Metropolitan Doha.[6] In 2009, the government launched the World Innovation Summit for Education (WISE), a global forum that brings together education stakeholders, opinion leaders and decision makers from all over the world to discuss educational issues.[134] The first edition was held in Doha in November 2009.[135] Some of the universities in Doha include: Carnegie Mellon University in Qatar Georgetown University School of Foreign Service in Qatar Hamad Bin Khalifa University Cornell University[136] HEC Paris Northwestern University in Qatar Texas A&M University at Qatar UCL Qatar[137] Virginia Commonwealth University Weill Cornell Medical College in Qatar Stenden University Qatar College of the North Atlantic Qatar University Qatar Faculty of Islamic Studies University of Calgary Sports[edit] Main article: Sport in Qatar Football[edit] Al Sadd is the most successful team in the Qatar Stars League See also: List of football stadiums in Qatar Football is the most popular sport in Doha. There are six Doha-based sports clubs with football teams currently competing in the Qatar Stars League, the country's top football league. They are Al Ahli, Al Arabi, Al Sadd, Al-Duhail and Qatar SC.[138] Al Sadd, Al Arabi and Qatar SC are the three most successful teams in the league's history.[139] Numerous football tournaments have been hosted in Doha. The most prestigious tournaments include the 1988 and 2011 editions of the AFC Asian Cup[140] and the 1995 FIFA World Youth Championship.[54] In December 2010, Qatar won the rights to host the 2022 FIFA World Cup.[141] Three of the nine newly announced stadiums will be constructed in Doha, including Sports City Stadium, Doha Port Stadium, and Qatar University Stadium. Additionally, the Khalifa International Stadium is set to undergo an expansion.[142] Considering the country's rapid development for 2022 World Cup, FIFA awarded the hosting rights of 2019 FIFA Club World Cup and 2020 FIFA Club World Cup also to Qatar.[143] Basketball[edit] Doha was the host of the official 2005 FIBA Asia Championship, where Qatar's national basketball team finished 3rd, its best performance to date, and subsequently qualified for the Basketball World Cup.[144] The majority of the teams that make up the official Qatari Basketball League are based in Doha. Volleyball[edit] Doha four times was the host of the official FIVB Volleyball Men's Club World Championship and three times host FIVB Volleyball Women's Club World Championship. Doha one time Host Asian Volleyball Championship.[145] Other sports[edit] Orry the Oryx, mascot of the 15th Asian Games, on the Doha Corniche in 2014 In 2001, Qatar became the first country in the Middle East to hold a women's tennis tournament with the inauguration of its Qatar Ladies Open tournament.[146] Doha also hosts International Tennis Federation (ITF) ladies tournaments. Since 2008, the Sony Ericsson Championships (equivalent to the ATP's season-ending Championships) has taken place in Doha, in the Khalifa International Tennis Complex, and features record prize money of $4.45 million, including a check of $1,485,000 for the winner, which represents the largest single guaranteed payout in women's tennis.[147] Doha hosted the 15th Asian Games, held in December 2006, spending a total of $2.8 billion for its preparation.[148] The city also hosted the 3rd West Asian Games in December 2005.[149] Doha was expected to host the 2011 Asian Indoor Games; but the Qatar Olympic Committee cancelled the event.[150] Powerboat races in Doha Bay The city submitted a bid for the 2016 Olympics.[151] On June 4, 2008, the city was eliminated from the shortlist for the 2016 Olympic Games. On August 26, 2011 it was confirmed that Doha would bid for the 2020 Summer Olympics.[152] Doha however failed to become a Candidate City for the 2020 Games.[153] The MotoGP motorcycling grand prix of Doha is held annually at Losail International Circuit, located just outside the city boundaries.[154] The city is also the location of the Grand Prix of Qatar for the F1 Powerboat World Championship, annually hosting a round in Doha Bay.[155] Beginning in November 2009, Doha has been host of The Oryx Cup World Championship, a hydroplane boat race in the H1 Unlimited season. The races take place in Doha Bay.[156] In April 2012 Doha was awarded both the 2014 FINA World Swimming Championships[157] and the 2012 World Squash Championships.[158] The fourth World Mindsports Championships took place in Doha from August 19 to August 27, 2017 with the participation of more than 1,000 competitors.[159] In 2014, Qatar was selected as the host of the 2019 World Athletics Championships, which is the seventeenth edition of the IAAF World Athletics Championships.[160] Doha won the bid to host the event over Barcelona and Eugene.[161] In 2020, Doha hosted the Qatar ExxonMobil Open, which received the Tournament of the Year award in the 250 category from the 2019 ATP Awards. The tournament won the award for the third time in five years.[162] Stadiums and sport complexes[edit] An indoor stadium in the Aspire Zone sporting complex Aspire Academy was launched in 2004 with the aim of creating world-class athletes. It is situated in the Doha Sports City Complex, which also accommodates the Khalifa International Stadium, the Hamad Aquatic Centre, the Aspire Tower and the Aspire Dome. The latter has hosted more than 50 sporting events since its inception, including some events during the 2006 Asian Games.[163] Sporting venues in Doha and its suburbs include: Hamad bin Khalifa Stadium – Al-Ahli Stadium Jassim Bin Hamad Stadium (Al Sadd Stadium) Al-Arabi Stadium – Grand Hamad Stadium Hamad Aquatic Centre Khalifa International Stadium – Main venue for the 2006 Asian Games. Khalifa International Tennis and Squash Complex Qatar Sports Club Stadium Culture[edit] Main article: Culture of Qatar Doha was chosen as the Arab Capital of Culture in 2010.[164] Cultural weeks organized by the Ministry of Culture, which featured both Arab and non-Arab cultures, were held in Doha from April to June to celebrate the city's selection.[165] Arts[edit] Main article: Qatari art Further information: Public art in Qatar and Collecting practices of the Al-Thani Family The five-storeyed Museum of Islamic Art designed by Pritzker Prize-winning architect I. M. Pei[166] The Museum of Islamic Art in Doha, opened in 2008, is regarded as one of the best museums in the region.[167] This, and several other Qatari museums located in the city, like the Arab Museum of Modern Art, falls under the Qatar Museums Authority (QMA) which is led by Sheikha Al-Mayassa bint Hamad bin Khalifa Al-Thani, the sister of the emir of Qatar.[168] The National Museum of Qatar, which was constructed in place of the original Qatar National Museum, opened to the public on 28 March 2019. Cinema[edit] Main article: Cinema of Qatar The Doha Film Institute (DFI) is an organisation established in 2010 to oversee film initiatives and create a sustainable film industry in Qatar. DFI was founded by H.E. Sheikha Al Mayassa bint Hamad bin Khalifa Al-Thani.[169] The Doha Tribeca Film Festival (DTFF), partnered with the American-based Tribeca Film Festival, was held annually in Doha from 2009 to 2012.[170] Media[edit] Main article: Media of Qatar See also: Television in Qatar Al Jazeera Arabic Building Qatar's first radio station, Mosque Radio, began broadcasting in the 1960s from Doha.[171] The multinational media conglomerate Al Jazeera Media Network is based in Doha with its wide variety of channels of which Al Jazeera Arabic, Al Jazeera English, Al Jazeera Documentary Channel, Al Jazeera Mubasher, beIN Sports Arabia and other operations are based in the TV Roundabout in the city.[172] Al-Kass Sports Channel's headquarters is also located in Doha.[173] Theatre[edit] Main article: Theatre in Qatar Theatre was introduced to Qatar in the mid-20th century. Theatrical performances are held at Qatar National Theater and at the Qatar National Convention Centre in Doha. International relations[edit] Algeirs, Algeria (since 2013)[174] Sarajevo, Bosnia and Herzegovina (since 2018)[175] Brasília, Brazil (since 2014)[176] Sofia, Bulgaria (since 2012)[177] Beijing, China (since 2008)[178] Alameda, California (since 2004)[179] San Salvador, El Salvador (since 2018)[180] Banjul, Gambia (since 2011)[181] Tbilisi, Georgia (since 2012)[182] Nur-Sultan, Kazakhstan (since 2011)[183] Bishkek, Kyrgyzstan (since 2018)[184] Port Louis, Mauritius (since 2007)[185] Mogadishu, Somalia (since 2014)[186] Tunis, Tunisia (since 1994)[187] Ankara, Turkey (since 2016)[188] Los Angeles, California, United States (since 2016)[189] Miami, Florida, United States (since 2016)[190] Libertador, Venezuela (since 2015)[191] Beit Sahour, Palestine (since 2009)[192] Gallery[edit] Click on the thumbnail to enlarge. Skyline of Doha West Bay from Sheraton Park. The spring festival at Souq Waqif, Doha An old mosque minaret stands out in front of the under-construction National Archive building in the Diwan Amiri Quarter of the Musheireb downtown Doha development. Qatar's Amir (ruler) is housed in the Amiri Diwan located in the historic Al Bidda district. These twin towers are among the earliest towers in Doha and serve as a great example of post-modern architecture. Msheireb Enrichment Centre moored off Doha Corniche is a learning center focused on the history and developments of Doha, particularly the Musheirib district. Aspire Park, Al Waab is one of the city's green spaces that forms a part of the Aspire zone. Doha skyline from the Museum of Islamic Art. Doha skyline at night. Doha Corniche is the 7 km long water front that connects the new district of West Bay with the old district of Al-Bidda and Al-Souq on the other end. Aerial view of a part of the city. The Katara cultural village is designed to be a hub of human interaction connecting theatre, literature, music, visual art, conventions and exhibitions in a planned development on the waterfront.[193] The post office building in Qatar sits located on the main Corniche street. See also[edit] Doha Declaration Doha Development Round of World Trade Organization (WTO) talks Qatar National Day which is held in Doha every year on December 18 References[edit] ^ "Doha municipality accounts for 40% of Qatar population". Gulf Times. 20 October 2015. Retrieved 23 October 2015..mw-parser-output cite.citation{font-style:inherit}.mw-parser-output .citation q{quotes:"\"""\"""'""'"}.mw-parser-output .id-lock-free a,.mw-parser-output .citation .cs1-lock-free a{background-image:url("//upload.wikimedia.org/wikipedia/commons/thumb/6/65/Lock-green.svg/9px-Lock-green.svg.png");background-image:linear-gradient(transparent,transparent),url("//upload.wikimedia.org/wikipedia/commons/6/65/Lock-green.svg");background-repeat:no-repeat;background-size:9px;background-position:right .1em center}.mw-parser-output .id-lock-limited a,.mw-parser-output .id-lock-registration a,.mw-parser-output .citation .cs1-lock-limited a,.mw-parser-output .citation .cs1-lock-registration a{background-image:url("//upload.wikimedia.org/wikipedia/commons/thumb/d/d6/Lock-gray-alt-2.svg/9px-Lock-gray-alt-2.svg.png");background-image:linear-gradient(transparent,transparent),url("//upload.wikimedia.org/wikipedia/commons/d/d6/Lock-gray-alt-2.svg");background-repeat:no-repeat;background-size:9px;background-position:right .1em center}.mw-parser-output .id-lock-subscription a,.mw-parser-output .citation .cs1-lock-subscription a{background-image:url("//upload.wikimedia.org/wikipedia/commons/thumb/a/aa/Lock-red-alt-2.svg/9px-Lock-red-alt-2.svg.png");background-image:linear-gradient(transparent,transparent),url("//upload.wikimedia.org/wikipedia/commons/a/aa/Lock-red-alt-2.svg");background-repeat:no-repeat;background-size:9px;background-position:right .1em center}.mw-parser-output .cs1-subscription,.mw-parser-output .cs1-registration{color:#555}.mw-parser-output .cs1-subscription span,.mw-parser-output .cs1-registration span{border-bottom:1px dotted;cursor:help}.mw-parser-output .cs1-ws-icon a{background-image:url("//upload.wikimedia.org/wikipedia/commons/thumb/4/4c/Wikisource-logo.svg/12px-Wikisource-logo.svg.png");background-image:linear-gradient(transparent,transparent),url("//upload.wikimedia.org/wikipedia/commons/4/4c/Wikisource-logo.svg");background-repeat:no-repeat;background-size:12px;background-position:right .1em center}.mw-parser-output code.cs1-code{color:inherit;background:inherit;border:inherit;padding:inherit}.mw-parser-output .cs1-hidden-error{display:none;font-size:100%}.mw-parser-output .cs1-visible-error{font-size:100%}.mw-parser-output .cs1-maint{display:none;color:#33aa33;margin-left:0.3em}.mw-parser-output .cs1-subscription,.mw-parser-output .cs1-registration,.mw-parser-output .cs1-format{font-size:95%}.mw-parser-output .cs1-kern-left,.mw-parser-output .cs1-kern-wl-left{padding-left:0.2em}.mw-parser-output .cs1-kern-right,.mw-parser-output .cs1-kern-wl-right{padding-right:0.2em}.mw-parser-output .citation .mw-selflink{font-weight:inherit} ^ Jump up to: a b c The Report: Qatar 2016. 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Retrieved 31 May 2018. ^ "Legal Framework". Embassy of Georgia to the State of Qatar. Retrieved 31 May 2018. ^ "زيارة الأمير الأخيرة لكازاخستان أعطت زخماً للعلاقات الثنائية" (in Arabic). Al Raya. 11 December 2015. Retrieved 31 May 2018. ^ "HH The Amir Issues Two Decrees". Government of the State of Qatar. 19 February 2018. Retrieved 31 May 2018. ^ "International Links". City Council of Port Louis. Retrieved 31 May 2018. ^ "Mungaab seeks Doha's help in reviving Mogadishu". Somali Agenda. 13 November 2014. Retrieved 31 May 2018. ^ "International Cooperation". Municipality of Tunis. Retrieved 31 May 2018. ^ "Doha, Ankara sign twinning agreement". Gulf Times. 24 August 2016. Retrieved 31 May 2018. ^ "Joint Statement by the United States and Qatar on the Conclusion of the Second Annual Economic and Investment Dialogue". U.S. Department of State. 13 December 2016. Retrieved 31 May 2018. ^ "Twinning Agreement between Miami and Doha". Istithmar USA. 5 June 2016. Retrieved 31 May 2018. ^ "HH the Emir, Venezuelan President Witness Signing of Agreements". Ministry of Foreign Affairs (Qatar). 25 November 2015. Retrieved 31 May 2018. ^ "Twinning". Beit Sahour Municipality Palestine. Archived from the original on 9 July 2018. Retrieved 30 May 2018. ^ "About Katara". Katara.net. Retrieved 2018-05-14. External links[edit] Wikimedia Commons has media related to Doha. Wikivoyage has a travel guide for Doha. 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See: Political status of Taiwan, Chagos Archipelago sovereignty dispute, Cyprus dispute, Status of Jerusalem, Artsakh-Azerbaijani conflict, Abkhaz-Georgian conflict and Georgian-Ossetian conflict showvteCapitals of Arab countriesAfricaAsia Algiers, Algeria Cairo, Egypt Djibouti, Djibouti El Aaiun (proclaimed) Tifariti (de facto), Sahrawi Arab Democratic Republic1 Hargeisa, Somaliland1 Khartoum, Sudan Mogadishu, Somalia Moroni, Comoros Nouakchott, Mauritania Rabat, Morocco Tripoli, Libya Tunis, Tunisia Abu Dhabi, United Arab Emirates Amman, Jordan Baghdad, Iraq Beirut, Lebanon Damascus, Syria Doha, Qatar Jerusalem (proclaimed) Ramallah (de facto), Palestine1 Kuwait City, Kuwait Manama, Bahrain Muscat, Oman Riyadh, Saudi Arabia Sana'a, Yemen 1 An unrecognised or partially-recognised nation showvteArab Capital of Culture Cairo 1996 (Egypt) Tunis 1997 (Tunisia) Sharjah 1998 (United Arab Emirates) Beirut 1999 (Lebanon) Riyadh 2000 (Saudi Arabia) Kuwait City 2001 (Kuwait) Amman 2002 (Jordan) Rabat 2003 (Morocco) Sanaʽa 2004 (Yemen) Khartoum 2005 (Sudan) Muscat 2006 (Oman) Algiers 2007 (Algeria) Damascus 2008 (Syria) Jerusalem 2009 (State of Palestine) Doha 2010 (Qatar) Sirte 2011 (Libya) Manama 2012 (Bahrain) Baghdad 2013 (Iraq) Tripoli 2014 (Libya) Constantine 2015 (Algeria) Sfax 2016 (Tunisia) showvteHost cities of Asian GamesSummer 1951: Delhi 1954: Manila 1958: Tokyo 1962: Jakarta 1966: Bangkok 1970: Bangkok 1974: Tehran 1978: Bangkok 1982: Delhi 1986: Seoul 1990: Beijing 1994: Hiroshima 1998: Bangkok 2002: Busan 2006: Doha 2010: Guangzhou 2014: Incheon 2018: Jakarta-Palembang 2022: Hangzhou Winter 1986: Sapporo 1990: Sapporo 1996: Harbin 1999: Kangwon 2003: Aomori 2007: Changchun 2011: Astana-Almaty 2017: Sapporo Authority control BNF: cb15084063c (data) GND: 4219005-8 LCCN: n81076793 MusicBrainz: 29aae758-4c4f-4d77-899c-1dcb67196b24 NKC: ge463353 SELIBR: 143052 VIAF: 136649381 WorldCat Identities: lccn-n81076793 <img src="//en.wikipedia.org/wiki/Special:CentralAutoLogin/start?type=1x1" alt="" title="" width="1" height="1" style="border: none; position: absolute;" /> Retrieved from "https://en.wikipedia.org/w/index.php?title=Doha&oldid=963332295" Categories: DohaCapitals in AsiaMunicipalities of QatarPopulated coastal places in QatarPopulated places established in 1825Populated places in QatarBurial sites of the House of Thani1825 establishments in AsiaHidden categories: CS1 maint: uses authors parameterCS1 Arabic-language sources (ar)Articles with short descriptionArticles containing Arabic-language textCoordinates on WikidataCommons category link is on WikidataWikipedia articles with BNF identifiersWikipedia articles with GND identifiersWikipedia articles with LCCN identifiersWikipedia articles with MusicBrainz area identifiersWikipedia articles with NKC identifiersWikipedia articles with SELIBR identifiersWikipedia articles with VIAF identifiersWikipedia articles with WorldCat identifiers Navigation menu Personal tools Not logged inTalkContributionsCreate accountLog in Namespaces ArticleTalk Variants Views ReadEditView history More Search Navigation Main pageContentsCurrent eventsRandom articleAbout WikipediaContact usDonateWikipedia store Contribute HelpCommunity portalRecent changesUpload file Tools What links hereRelated changesUpload fileSpecial pagesPermanent linkPage information
  13. Jul 2018
    1. On 2014 Dec 15, Ivan Shatsky commented:

      General comment: The phenomenon studied in this paper is very exciting. I read the article with a great interest. Unfortunately, I regret to say that the underlying mechanism remained uncovered. Although I agree that the authors identified some curious structures within the 5’UTRs of HOXA mRNAs which might be implicated in the regulation of these mRNAs by RPL38, I did not find sufficient evidence for existence of IRES-elements in these mRNAs. As in numerous other similar investigations, to identify IRES-elements the authors employed the method of DNA bicistronic constructs, the approach that had been repeatedly shown to be associated with almost unavoidable artifacts (see Jackson Cold Spring Harb Perspect Biol. 2013 Feb 1;5(2); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90.). And I suspect this paper is not free of those artifacts either (see below). Several crucial control experiments necessary to support or to exclude the IRES-mediated mechanism have been recently described (for references see Shatsky et al. Mol Cells. 2010 Oct; 30(4):285-93). One of them, for instance, is the ratio of translational activities for m7G capped versus uncapped (A-capped) monocistronic constructs. This value estimates contribution of the cap to the translational potential of a 5’UTR under selected conditions. If this contribution is very high (as is the case of cap-dependent mRNAs) one may exclude the presence of a true IRES. I think that this and other obligatory controls are feasible to perform with cells C3H10T1/2 used in this paper but they were not done.

      Some specific points:

      1. The authors regard the cap-independent and IRES-dependent modes of translation initiation as synonymous mechanisms and support this notion with reference 21. I should stress that not all specialists in eukaryotic translation would share this opinion since nobody has ever shown that a 5’end dependent translation initiation cannot be regulated by specific structures within the respective 5' UTR. The opposite has recently been demonstrated (Terenin et al. Nucleic Acids Res. 2013 Feb 1;41(3):1807-16.)
      2. When listing examples of cellular IRESs identified to date (second paragraph), the authors mention c-myc, Apaf-1, XIAP. To the best of my knowledge, these cellular IRESs have been disproved (Andreev et al. Nucleic Acids Res. 2009 Oct;37(18):6135-47; Bert et al. RNA. 2006 Jun;12(6):1074-83; Baranick et al. Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4733-8; van Eden et al. RNA. 2004 Apr;10(4):720-30); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90. )
      3. “Extended data Figure 1a” raises a great concern: the HCV IRES should not be used as a normalizing construct for testing bicistronic DNAs since the HCV IRES has been reported to harbor a cryptic promoter (Dumas, E. et al. 2003 Nucleic Acids Res. 31 (4): 1275-1281) and hence may produce capped monocistronic mRNAs . By the way, among viral IRESs characterized to date the HCV IRES is regarded as one of the weakest.
      4. The control test with Rluc shRNA (Extended data Figure 1 b,c) strongly suggests that some significant amount of monocistronic (and therefore capped) Fluc mRNAs is present in transfected cells since the residual Fluc activity after RNA interference is too high. This may also be the case for the control bicistronic mRNA containing the HCV IRES (see point 3). Otherwise, the pictures 1b and 1c must be similar. At least, in the analogous test performed in our lab, the Rluc and Fluc activities fall down to the similar background levels (Fig. 2D in Dmitriev et al. Mol Cell Biol. 2007 Jul;27(13):4685-97).
      5. The authors suggest that the IRES elements are mostly confined within ~300 nts proximal to the start codon. As a support to this conclusion, they note that some of HOXA mRNAs possess a 5’ UTR of that size. If so, why the activity of HOXA9 construct 944-1266 is much lower than that for the full length 5’UTR (Fig. 1d)?<br>
      6. “Extended Figure 1d”. This control is useless. It only shows that the bicistronic mRNA of the expected size is present in transfected cells but unable to show the presence of monocistronic mRNAs starting within the intercistronic region. The corresponding bands won’t be seen.
      7. On the base of pull-down experiments the authors claim that 80S ribosomes are specifically formed on their IRES-elements. The problem is that they use 10mM of magnesium in these expts, i.e. the concentration at which the assembly of translation initiation complexes in mammalian systems should not occur.
      8. The mode of action of TIE element looks absolutely puzzling. It is even difficult to imagine any mechanism for its operation. My question: is it specific to these particular cells?


      This comment, imported by Hypothesis from PubMed Commons, is licensed under CC BY.

  14. Feb 2018
    1. On 2014 Dec 15, Ivan Shatsky commented:

      General comment: The phenomenon studied in this paper is very exciting. I read the article with a great interest. Unfortunately, I regret to say that the underlying mechanism remained uncovered. Although I agree that the authors identified some curious structures within the 5’UTRs of HOXA mRNAs which might be implicated in the regulation of these mRNAs by RPL38, I did not find sufficient evidence for existence of IRES-elements in these mRNAs. As in numerous other similar investigations, to identify IRES-elements the authors employed the method of DNA bicistronic constructs, the approach that had been repeatedly shown to be associated with almost unavoidable artifacts (see Jackson Cold Spring Harb Perspect Biol. 2013 Feb 1;5(2); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90.). And I suspect this paper is not free of those artifacts either (see below). Several crucial control experiments necessary to support or to exclude the IRES-mediated mechanism have been recently described (for references see Shatsky et al. Mol Cells. 2010 Oct; 30(4):285-93). One of them, for instance, is the ratio of translational activities for m7G capped versus uncapped (A-capped) monocistronic constructs. This value estimates contribution of the cap to the translational potential of a 5’UTR under selected conditions. If this contribution is very high (as is the case of cap-dependent mRNAs) one may exclude the presence of a true IRES. I think that this and other obligatory controls are feasible to perform with cells C3H10T1/2 used in this paper but they were not done.

      Some specific points:

      1. The authors regard the cap-independent and IRES-dependent modes of translation initiation as synonymous mechanisms and support this notion with reference 21. I should stress that not all specialists in eukaryotic translation would share this opinion since nobody has ever shown that a 5’end dependent translation initiation cannot be regulated by specific structures within the respective 5' UTR. The opposite has recently been demonstrated (Terenin et al. Nucleic Acids Res. 2013 Feb 1;41(3):1807-16.)
      2. When listing examples of cellular IRESs identified to date (second paragraph), the authors mention c-myc, Apaf-1, XIAP. To the best of my knowledge, these cellular IRESs have been disproved (Andreev et al. Nucleic Acids Res. 2009 Oct;37(18):6135-47; Bert et al. RNA. 2006 Jun;12(6):1074-83; Baranick et al. Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4733-8; van Eden et al. RNA. 2004 Apr;10(4):720-30); Lemp et al. Nucleic Acids Res. 2012 Aug;40(15):7280-90. )
      3. “Extended data Figure 1a” raises a great concern: the HCV IRES should not be used as a normalizing construct for testing bicistronic DNAs since the HCV IRES has been reported to harbor a cryptic promoter (Dumas, E. et al. 2003 Nucleic Acids Res. 31 (4): 1275-1281) and hence may produce capped monocistronic mRNAs . By the way, among viral IRESs characterized to date the HCV IRES is regarded as one of the weakest.
      4. The control test with Rluc shRNA (Extended data Figure 1 b,c) strongly suggests that some significant amount of monocistronic (and therefore capped) Fluc mRNAs is present in transfected cells since the residual Fluc activity after RNA interference is too high. This may also be the case for the control bicistronic mRNA containing the HCV IRES (see point 3). Otherwise, the pictures 1b and 1c must be similar. At least, in the analogous test performed in our lab, the Rluc and Fluc activities fall down to the similar background levels (Fig. 2D in Dmitriev et al. Mol Cell Biol. 2007 Jul;27(13):4685-97).
      5. The authors suggest that the IRES elements are mostly confined within ~300 nts proximal to the start codon. As a support to this conclusion, they note that some of HOXA mRNAs possess a 5’ UTR of that size. If so, why the activity of HOXA9 construct 944-1266 is much lower than that for the full length 5’UTR (Fig. 1d)?<br>
      6. “Extended Figure 1d”. This control is useless. It only shows that the bicistronic mRNA of the expected size is present in transfected cells but unable to show the presence of monocistronic mRNAs starting within the intercistronic region. The corresponding bands won’t be seen.
      7. On the base of pull-down experiments the authors claim that 80S ribosomes are specifically formed on their IRES-elements. The problem is that they use 10mM of magnesium in these expts, i.e. the concentration at which the assembly of translation initiation complexes in mammalian systems should not occur.
      8. The mode of action of TIE element looks absolutely puzzling. It is even difficult to imagine any mechanism for its operation. My question: is it specific to these particular cells?


      This comment, imported by Hypothesis from PubMed Commons, is licensed under CC BY.